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Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)–positive epithelial malignancy with an extensive inflammatory infiltrate. Traditional RNA-sequencing techniques uncovered only microenvironment signatures, while the gene expression of the tumor epithelial compartment has remained a mystery. Here, we use Smart-3SEQ to prepare transcriptome-wide gene expression profiles from microdissected NPC tumors, dysplasia, and normal controls. We describe changes in biological pathways across the normal to tumor spectrum and show that fibroblast growth factor (FGF) ligands are overexpressed in NPC tumors, while negative regulators of FGF signaling, including SPRY1, SPRY2, and LGALS3, are down-regulated early in carcinogenesis. Within the NF-κB signaling pathway, the critical noncanonical transcription factors, RELB and NFKB2, are enriched in the majority of NPC tumors. We confirm the responsiveness of EBV-positive NPC cell lines to targeted inhibition of these pathways, reflecting the heterogeneity in NPC patient tumors. Our data comprehensively describe the gene expression landscape of NPC and unravel the mysteries of receptor tyrosine kinase and NF-κB pathways in NPC.

Epstein-Barr virus (EBV) infection is strongly associated with nasopharyngeal carcinoma, a common cancer in Southeast Asia and certain regions of Africa. However, the dynamics of EBV episome maintenance in infected nasopharyngeal epithelial (NPE) cells remain largely undefined. Here, we report the establishment of a highly efficient cell-free EBV infection method for NPE cells. By using this method, we have defined some of the dynamic events involved in the early stage of EBV infection in NPE cells. We report, for the first time, a rapid loss of EBV copies from infected NPE cells during the first 12-72 h post-infection. The rate of EBV loss slowed at later stages of infection. Live cell imaging revealed that the freshly infected NPE cells were delayed in entry into mitosis compared with uninfected cells. Freshly infected NPE cells transcribed significantly higher levels of lytic EBV genes BZLF1 and BMRF1 yet significantly lower levels of EBER1/2 than stably infected NPE cells. Notably, there were very low or undetectable levels of protein expressions of EBNA1, LMP1, Zta and Rta in freshly infected NPE cells, whereas EBNA1 and LMP1 proteins were readily detected in stable EBV-infected NPE cells. The kinetics of EBV loss and the differential EBV gene expression profiles between freshly and stably infected NPE cells are in line with the suggestion of epigenetic changes in the EBV genome that affect viral gene expression and the adaptation of host cells to EBV infection to maintain persistent EBV infection in NPE cells.

EBV infection of preinvasive nasopharyngeal epithelium is believed to be an initiation step during pathogenesis of nasopharyngeal carcinoma (NPC), but the mechanisms remain poorly understood. Here we report a novel mechanism driving NPC metastasis through the EBV-encoded LMP1-mediated metabolic reprogramming, via activation of IGF1-mTORC2 signaling and nuclear acetylation of the Snail promoter by the PDHE1α, an enzyme involved in glucose metabolism. Mechanistically, EBV-LMP1 increases the cellular secretion of IGF1 which promotes phosphorylation of IGF1R to activate mTORC2/AKT signaling linking glucose metabolism to cell motility. LMP1 expression facilitates translocation of mitochondrial PDHE1α into the nucleus in a phosphorylation-dependent manner at Ser293 residue. Functionally, nuclear PDHE1α promotes H3K9 acetylation on the Snail promoter to enhance cell motility, thereby driving cancer metastasis. Importantly, the IGF1/mTORC2/PDHE1α/Snail axis correlates significantly with disease progression and poor prognosis in NPC patients. This study highlights the functional importance of IGF1-mTORC2-PDHE1α signaling mediated by EBV-LMP1 in NPC pathogenesis.

Background: NPC is particularly common in Southern China and Southeast Asia. The standard treatment for NPC is radiotherapy with or without adjuvant chemotherapy. Despite the high 5-year overall survival rate (85.6%) in early-stage patients, 15% to 20% of the patients still experience recurrence or metastasis. The development of new targeted therapy on NPC is still lagging behind. Considered that phase I clinical trials often recruit late-stage patients who previously received multiple treatments, lack of drug-resistant models may be one of the possible hindrances in targeted therapy development. In-vivo preclinical NPC models are very limited for research. Given the close association with Epstein-Barr virus (EBV) infection in NPC, only four EBV positive (EBV +ve) PDXs and one EBV +ve cell line are available for research. Our lab has been engaging in establishing new NPC models. Previously, we had successfully established four new EBV +ve PDXs: Xeno23, Xeno32, Xeno47 and Xeno76. To establish a more clinically relevant models for preclinical studies and drug resistance research, we continuously treat the PDX transplanted mice with cisplatin. And we successfully established the first cisplatin-resistant Xeno76 (Xeno76-CR) NPC PDX. Method: A 1 mm3 PDX tissues were implanted subcutaneously in Nude mice. Once the tissue started growing into ~100 mm3 in size, 3-5 mg/kg cisplatin was injected intraperitoneally weekly. Once the end point has met, the tumor tissue was repeatedly implanted into another mice, while the rest of the tissues were used for further evaluation. Result: The growth rate of Xeno76-CR has no significant difference with Xeno76 in Nude mice. We confirmed that Xeno76-CR is resistant to cisplatin in-vivo. When treating both Xeno76 and Xeno76-CR with 3 mg/kg cisplatin weekly, there is statistically significant 85.4% tumor growth inhibition in Xeno76, but no significant inhibition effect in cisplatin-treated Xeno76-CR. EBV infection of Xeno76-CR was confirmed with EBER staining. We have also studied whether Xeno76-CR is drug- or specifically cisplatin-resistant. We tested the Xeno76-CR with ixazomib, an oral proteasome inhibitor that can inhibit nuclear factor-kB (NF-kB) signalling pathway, and found significant 57% tumor growth inhibition in-vivo. Conclusion: We have established the first and only EBV +ve cisplatin-resistant NPC PDX, Xeno76-CR, that is a valuable and clinically relevant model for targeted therapies preclinical studies and resistant research. Single-cell RNA sequencing will be performed to examine if stem cell plays any role in cisplatin-resistance. Further genome and transcriptome expression profile and characterization studies are still ongoing. Citation Format: Ka Yee Li, Katie Sze Wai Fung, Chanping You, Yim Ling Yip, Victor Ho Fun Lee, George Sai Wah Tsao. Establishment and characterization of cisplatin-resistant nasopharyngeal carcinoma (NPC) patient-derived xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6021.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival., Nasopharyngeal carcinoma (NPC) lacks effective diagnostic and therapeutic strategies, in particular at advanced stages. Here, the authors show that expression of the somatostatin receptor 2 is induced by Epstein-Barr virus in NPC and has a key role in the diagnosis, imaging, targeted therapies and prognosis of NPC.

Establishment of continuous cell lines and patient-derived xenografts (PDXs) remains a major challenge for nasopharyngeal carcinoma (NPC). All the reported NPC cell lines except one (C666-1) lost their Epstein–Barr virus (EBV) episomes upon long-term propagation. Furthermore, many commonly used NPC cell lines are contaminated with genetic elements of HeLa. By inclusion of a Rho kinase inhibitor (Y-27632) in the growth medium to suppress lytic reactivation of EBV, we have established two new NPC cell lines (C17 and NPC43) that retain EBV episomes. The low rate of NPC PDX establishment may reflect an NPC cell growth dependency on the tumor microenvironment. Nonetheless, we have established and characterized several new NPC PDXs for NPC investigation. Telomerase expression, in combination with the common genetic alterations present in NPC, efficiently immortalized primary cultures of nonmalignant nasopharyngeal epithelial cells. The lessons learnt from the establishment of these cell lines and NPC PDXs will be discussed.

Using Epstein‐Barr virus (EBV)‐induced cancer cells and HeLa cells as a comparative study model, a novel and safe dual‐EBV‐oncoproteins‐targeting pH‐responsive peptide engineering, coating, and guiding approach to achieve precision targeting and treatment strategy against EBV‐associated cancers is introduced. Individual functional peptide sequences that specifically bind to two overexpressed EBV‐specific oncoproteins, EBNA1 (a latent cellular protein) and LMP1 (a transmembrane protein), are engineered in three different ways and incorporated with a pH‐sensitive tumor microenvironment (TME)‐cleavable linker onto the upconversion nanoparticles (UCNP) NaGdF4:Yb3+, Er3+@NaGdF4 (UCNP‐Pn, n = 5, 6, and 7). A synergistic combination of the transmembrane LMP1 targeting ability and the pH responsiveness of UCNP‐Pn is found to give specific cancer differentiation with higher cellular uptake and accumulation in EBV‐infected cells, thus a lower dose is needed and the side effects and health risks from treatment would be greatly reduced. It also gives responsive UC signal enhancement upon targeted dual‐protein binding and shows efficacious EBV cancer inhibition in vitro and in vivo. This is the first example of simultaneous imaging and inhibition of two EBV latent proteins, and serves as a blueprint for next‐generation peptide‐guided precision delivery nanosystem for the safe monitoring and treatment against one specific cancer., The upconversion nanoplatform with desired responsiveness toward tumor microenvironment is introduced. Thereafter, EBV‐cancer model is used to investigate by utilizing the safe nanoplatform via targeting LMP1 and EBNA1 with responsive emission, satisfactory prohibitive performances are observed in vitro and in vivo, thanks to the specific dual‐targeting peptides. This proposed multifunctional nanoplatform can achieve both monitoring and treatment for EBV cancer.

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.

NF-κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF-κB is common in NPC and may contribute to NPC development. The role of NF-κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF-κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF-κB signaling is a common phenotype in telomerase-immortalized NPE cell lines. Our results suggest that NF-κB activation promotes the growth of telomerase-immortalized NPE cells, and suppression of NF-κB activity inhibits their proliferation. Furthermore, we observed upregulation of c-Myc, IL-6 and Bmi-1 in our immortalized NPE cells. Inhibition of NF-κB downregulated expression of c-Myc, IL-6 and Bmi-1, suggesting that they are downstream events of NF-κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF-κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF-κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.

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.

Accumulating evidence indicates that oncogenic viral protein plays a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a transmembrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-κB is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-κB signaling to mediate aerobic glycolysis. NF-κB activation is involved in the LMP1-induced upregulation of glucose transporter 1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-κB activation and Glut-1 transcription. Interfering NF-κB signaling had no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that the Glut-1 gene is a direct target gene of NF-κB signaling. Furthermore, we demonstrated that C-terminal activating region 2 (CTAR2) of LMP1 is the key domain involved in mTORC1 activation, mainly through IKKβ-mediated phosphorylation of TSC2 at Ser 939 . Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-κB/Glut-1 represents a novel therapeutic target against NPC. IMPORTANCE Aerobic glycolysis is one of the hallmarks of cancer, including NPC. Recent studies suggest a role for LMP1 in mediating aerobic glycolysis. LMP1 expression is common in NPC. The delineation of essential signaling pathways induced by LMP1 in aerobic glycolysis contributes to the understanding of NPC pathogenesis. This study provides evidence that LMP1 upregulates Glut-1 transcription to control aerobic glycolysis and tumorigenic growth of NPC cells through mTORC1/NF-κB signaling. Our results reveal novel therapeutic targets against the mTORC1/NF-κB/Glut-1 signaling axis in the treatment of EBV-infected NPC.

Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression.

Nasopharyngeal carcinoma (NPC) is a common disease among southern Chinese. The major etiological factors proposed for NPC pathogenesis include genetic susceptibility, environment factors and EBV infection. In the high risk population, genetic susceptibility to NPC has been mapped to the HLA loci and adjacent genes in MHC region on chromosome 6p21. Consumption of preserved food including salted fish has been implicated in its etiology in earlier studies. Its contribution to pathogenesis of NPC remains to be determined. A decreasing trend of NPC incidence was observed in Hong Kong, Taiwan and Singapore in recent years which may be accounted by a change of dietary habits. A comprehensive epidemiological study will help to elucidate the relative importance of various risk factors in the pathogenesis of NPC. Despite the close association of EBV infection with NPC, the etiological role of EBV in NPC pathogenesis remains enigmatic. EBV infection in primary nasopharyngeal epithelial cells is uncommon and difficult to achieve. EBV does not transform primary nasopharyngeal epithelial cells into proliferative clones, which contrasts greatly with the well-documented ability of EBV to transform and immortalize primary B cells. Genetic alterations identified in premalignant nasopharyngeal epithelium may play crucial roles to support stable EBV infection. Subsequently, latent and lytic EBV gene products may drive clonal expansion and transformation of premalignant nasopharyngeal epithelial cells into cancer cells. Stromal inflammation in nasopharyngeal mucosa is believed to play an important role in modulating the growth and possibly drive the malignant transformation of EBV-infected nasopharyngeal epithelial cells. Furthermore, there are increasing evidences supporting a role of EBV infection to evade host immune surveillance. EBV-infected cells may have selective growth advantages in vivo by acquiring a stress-resistance phenotype. Understanding the etiological factors and pathogenesis of NPC will contribute effectively to the prevention and treatment of this disease.

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.

// Su Hang 1, 2 , Agnes F.Y. Tiwari 1 , Hextan Y.S. Ngan 3 , Yim-Ling Yip 4 , Annie L.M. Cheung 4 , Sai Wah Tsao 4 , Wen Deng 1 1 School of Nursing, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China 2 College of Forensic Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shanxi Province, P.R. China 3 Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, SAR, China 4 School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China Correspondence to: Wen Deng, email: wdeng@hku.hk Keywords: cervical epithelial cells, immortalization, p16INK4a, telomerase, HPV-negative Received: March 13, 2016 Accepted: May 29, 2016 Published: June 17, 2016 ABSTRACT Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16 INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16 INK4a , which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16 INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer.

Undifferentiated nasopharyngeal carcinomas (NPCs) are commonly present with latent EBV infection. However, events regulating EBV infection at early stages of the disease and the role of EBV in disease pathogenesis are largely undefined. Genetic alterations leading to activation of cyclin D1 signaling in premalignant nasopharyngeal epithelial (NPE) cells have been postulated to predispose cells to EBV infection. We previously reported that loss of p16, a negative regulator of cyclin D1 signaling, is a frequent feature of NPC tumors. Here, we report that early premalignant lesions of nasopharyngeal epithelium overexpress cyclin D1. Furthermore, overexpression of cyclin D1 is closely associated with EBV infection. Therefore we investigated the potential role of cyclin D1 overexpression in dysplastic NPE cells in vitro. In human telomerase reverse transcriptase-immortalized NPE cells, overexpression of cyclin D1 or a p16-resistant form of CDK4 (CDK4(R24C)) suppressed differentiation. This suppression may have implications for the close association of EBV infection with undifferentiated NPC. In these in vitro models, we found that cellular growth arrest and senescence occurred in EBV-infected cell populations immediately after infection. Nevertheless, overexpression of cyclin D1 or a p16-resistant form of CDK4 or knockdown of p16 in the human telomerase reverse transcriptase-immortalized NPE cell lines could counteract the EBV-induced growth arrest and senescence. We conclude that dysregulated expression of cyclin D1 in NPE cells may contribute to NPC pathogenesis by enabling persistent infection of EBV., published_or_final_version

Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ΔNp63α is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ΔNp63α in cell cycle regulation. ΔNp63α accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ΔNp63α knockdown promoted mitotic exit. ΔNp63α shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ΔNp63α. Our results suggest that ΔNp63α level is cell cycle-regulated and may play a role in the regulation of mitotic exit.

Cell immortalization is regarded as an early and pre-requisite step in tumor development. Defining the specific genetic events involved in cell immortalization may provide insights into the early events of carcinogenesis. Nasopharyngeal carcinoma is common among the Southern Chinese population. Epstein-Barr virus (EBV) infection is associated closely with nasopharyngeal carcinoma. The involvement of LMP1 (an EBV-encoded oncogene) has been implicated in the pathogenesis of nasopharyngeal carcinoma. In this study, LMP1 expression, in combination with ectopic expression of hTERT (catalytic unit of human telomerase), was shown to extend the life span of primary cultures of nasopharyngeal epithelial cells and facilitate the immortalization of one of the cell lines (NP446). This is the first report on the successful immortalization of nasopharyngeal epithelial cells involving LMP1. The events associated with the immortalization of nasopharyngeal epithelial cells by LMP1/hTERT were characterized. Expression of c-Myc, Bmi-1, and Id-1 were upregulated at an early stage of immortalization. At a later stage of immortalization, downregulation of p21 and p16 expression were observed. Upregulation of EGFR expression and activation of MAPK signaling pathway were observed in LMP1/hTERT-immortalized nasopharyngeal epithelial cells. The LMP1/hTERT-immortalized NP446 cells were non-tumorigenic in immunosuppressed nude mice and retained anchorage-dependent growth, suggesting that additional events are required for tumorigenic transformation. The ability of the EBV-encoded LMP1, in the presence of hTERT expression, to extend the life span and immortalize primary cultures of nasopharyngeal epithelial cells supports the involvement of EBV infection and its viral products in the early stage of pathogenesis of nasopharyngeal carcinoma.

How oncogenic virus rewires glucose flux to regulate cell motility is not well-understood. Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis. Here we demonstrate that EBV-encoded LMP1 enhances glycolysis thereby accelerating cell mobility of nasopharyngeal epithelial (NPE) cells. Activation of IGF1/mTORC2/AKT/PDHE1α signaling which mediates Histone 3 acetylation is involved in cell motility in EBV-infected NPE cells. Blocking the glucose uptake by pharmacological inhibitors effectively suppress glycolysis as well as the cell motility, linking the glucose metabolism to cell movement. We further show that mTORC2/AKT mediated phosphorylation of pyruvate decarboxylase kinase 1 (PDHK1) and its substrate, pyruvate decarboxylase E1α (PDHE1α) component plays a significant role in cell motility. PDHE1α knockdown effectively blocks LMP1-induced cell motility while overexpression of its phosphomimetic mutant S293A but not S293D inhibits LMP1-induced cell motility, supporting the involvement of PDHE1α in enhanced cell motility is depended on Ser293 phosphorylation. Furthermore, PDHE1α translocation into nucleus enhances Histone 3 acetylation and its binding to the promoter region of snail to enhance cell motility. Finally, we find that LMP1 increases the mRNA expression and the secretion of the ligand IGF1, which promotes phosphorylation of IGF1R as the key regulator for LMP1-induced mTORC2/AKT activation. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells to promote cell motility involving activation of mTORC2/AKT/PDHE1α. 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, Wing Chong Tang, Yim Ling Yip, Wen Deng, Lin Jia, Sai Wah Tsao. Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility 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 3079.

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.

The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/CCdh1-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells. © 2008 by The American Society for Cell Biology., published_or_final_version

NF-κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF-κB is common in NPC and may contribute to NPC development. The role of NF-κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF-κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF-κB signaling is a common phenotype in telomerase-immortalized NPE cell lines. Our results suggest that NF-κB activation promotes the growth of telomerase-immortalized NPE cells, and suppression of NF-κB activity inhibits their proliferation. Furthermore, we observed upregulation of c-Myc, IL-6 and Bmi-1 in our immortalized NPE cells. Inhibition of NF-κB downregulated expression of c-Myc, IL-6 and Bmi-1, suggesting that they are downstream events of NF-κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF-κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF-κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21., published_or_final_version

Accumulating evidence indicates that oncogenic viral protein exerts a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a trans-membrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-κB is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-κB signaling to mediate aerobic glycolysis. NF-κB activation is involved for LMP1-induced upregulation of glucose transptor-1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Collectively, blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-κB activation and Glut-1 transcription. Interfering NF-κB signaling has no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that Glut-1 is a direct target gene of NF-κB signaling. Furthermore, we demonstrated that the LMP1 C-terminal activating region (CTAR) 2 is the key domain involved in mTORC1 activation, mainly through IKKβ-mediated phosphorylation of TSC2 at Ser939. Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-κB/Glut-1 represents a novel therapeutic target against NPC. Acknowledgement: 96 800x600 This project was supported by the General Research Fund (HKU 779810M, 17120814 and 17161116), CRF equipment grant (1061402980, Health and Medical Research Fund of Hong Kong (12110782), AoE grant (AoE/M-06/08) and TBRS grant (T12-401/13-R). We thank Prof. Dongyan Jin (Department of Biochemistry, The University of Hong Kong) for the kind gifts of IKK related plasmids and Prof. Zhenguo Wu (Division of Life Science, The Hong Kong University of Science and Technology) for the discussion and interpretation of the data. We also thank Mr. Tony Chan for his technical support. Note: This abstract was not presented at the meeting. Citation Format: George S. Tsao, Jun Zhang, Lin Xia, C Tsang, Weitao Lin, Y Yip, W Deng, K Lo, M Lung. The role of EBV infection in aerobic glycolysis in 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 4417. doi:10.1158/1538-7445.AM2017-4417

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.

BACKGROUND: Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. METHODS: In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. RESULTS: Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. CONCLUSIONS: Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC., published_or_final_version

Nasopharyngeal carcinoma (NPC) is etiologically associated with Epstein-Barr virus (EBV) infection. However, the exact role of EBV in NPC pathogenesis remains elusive. Activation of signal transducer and activator of transcription 3 (STAT3) is common in human cancers including NPC and plays an important role in the pathogenesis and progression of human cancers. Interleukin-6 (IL-6), a major inflammatory cytokine, is a potent activator of STAT3. In this study, we report that EBV-infected immortalized nasopharyngeal epithelial (NPE) cells often acquire an enhanced response to IL-6-induced STAT3 activation to promote their growth and invasive properties. Interestingly, this enhanced IL-6/STAT3 response was mediated by overexpression of IL-6 receptor (IL-6R). Furthermore, IL-6R overexpression enhanced IL-6-induced STAT3 activation in uninfected immortalized NPE cells in vitro, and promoted growth and tumorigenicity of EBV-positive NPC cell line (C666-1) in vivo. Moreover, it is shown for the first time that IL-6R was overexpressed in clinical specimens of NPC. IL-6 expression could also be strongly detected in the stromal cells of NPC and a higher circulating level of IL-6 was found in the sera of advance-staged NPC patients compared to the control subjects. Therefore, IL-6R overexpression, coupled with enhanced IL-6/STAT3 signaling may facilitate the malignant transformation of EBV-infected premalignant NPE cells into cancer cells, and enhance malignant properties of NPC cells. © 2013 Zhang et al., published_or_final_version

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development. © 2012 Deng et al., published_or_final_version

Epithelial-mesenchymal transition (EMT), characterized by cadherin switching, contributes to cancer metastasis. Our recent study showed that Id-1 (inhibitor of differentiation-1) promotes metastasis in esophageal cancer cells, but whether the invasive and metastatic dynamics can be induced early in the carcinogenesis process is still unclear. Immortalization is regarded as the initial stage in the malignant transformation of normal cells. In this study, we investigated the role and mechanisms of Id-1 in inducing EMT and cell invasiveness in immortalized esophageal epithelial cells. We found that immortalized epithelial cells expressed higher endogenous levels of Id-1 compared with normal cells. Ectopic Id-1 expression inhibited the differentiation of immortalized esophageal epithelial cells and promoted cadherin switching, which was accompanied by increased adhesiveness to extracellular matrix, cell motility, migratory potential and matrix metalloproteinase-dependent invasiveness. GTPase activity assays showed that over-expression or short-hairpin RNA knockdown of Id-1 led to corresponding changes in Rac1 activity, whereas RhoA activity was significantly decreased with Id-1 depletion. Inhibitors targeting Rac1, RhoA, and Rho kinase suppressed the invasiveness of Id-1-expressing NE2-hTERT cells. Knockdown of N-cadherin in Id-1-over-expressing cells inhibited cell invasiveness and down-regulated RhoA activity. These data suggest that the Id-1-induced invasive potential may be regulated through the N-cadherin-RhoA axis and Rac1 activation.

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

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.

Nasopharyngeal carcinoma (NPC) is a common cancer among Southern Chinese. Many molecular and cellular events, particularly those occurring at the early stage of the NPC development, have not been well defined. Immortalization is a pre-requisite property of cancer cells and is regarded as an early event in human carcinogenesis. Defining and understanding these molecular and cellular events could provide us important insights to the early events involved in the carcinogenesis process. Our lab has established multiple immortalized nasopharyngeal epithelial (NP) cell lines using telomerase alone or in combination with other genetic elements including Bmi-1, cyclin D1 and knocking down p16. We observed that NF-Kappa B are commonly activated in immortalized cells compared to primary cells. Immunofluorescence staining and western blotting assay revealed activation of canonical NF-kappa B pathway in these immortalized nasopharyngeal epithelial cell lines with nuclear translocation of NF-kappa B p65 subunit. Interestingly, EGFR is also commonly expressed in our immortalized nasopharyngeal epithelial cells and may contribute to the constitutive activation of NF-kappa B in immortalized nasopharyngeal epithelial cells. Noteworthy, Epstein - Barr Virus (EBV) infection is closely associated with NPC development. There are studies reporting that activation of NF-kappa B and overexpression of EGFR are derived by EBV encoded LMP1 in NPC (Miller et al. Oncogene. 16, 1998). Our data also show LMP1 could induce NF-kappa B activation and EGFR overexpression in NPC cell line. Thus, the activation of NF-kappa B and overexpression of EGFR may be an important component of EBV infection in epithelial cells and contributing to the development of NPC. We further showed that activation of NF-kappa B signaling appears to be essential for the survival of immortalized nasopharyngeal epithelial cells as inhibition of NF-kappa B activity by a specific inhibitor (Nemo binding domain peptide), could selectively inhibit growth of the immortalized nasopharyngeal epithelial cells but not in primary nasopharyngeal epithelial cells. To study the functional interaction of NF-kappa B activation with EGFR, we have examined if EGFR overexpression may support immortalization of nasopharyngeal epithelial cells. We observed that functional EGFR could facilitate cell growth and protect cells from apoptosis. We are now exploring if activation of NF-kappa B and EGFR overexpression may be functionally related during immortalization of nasopharyngeal epithelial cells appears. Citation Format: Dandan Zhu, Yim Ling Yip, Wen Deng, Chi Man Tsang, Lai Man Cheung, Sai Wah Tsao. The potential role of NF-κB activation in the immortalization of nasopharyngeal epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1300. doi:10.1158/1538-7445.AM2013-1300 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Poorly or undifferentiated nasopharyngeal carcinoma (NPC) is regularly associated with Epstein-Barr virus (EBV) infection. EBV infection of premalignant nasopharyngeal epithelial cells has been postulated to play an important role in the pathogenesis of nasopharyngeal carcinoma. We have previously reported that genetic alterations could be detected in premalignant nasopharyngeal epithelial tissues. It is not clear if these genetic alterations may associate with the establishment of EBV infection. In this study, we have examined the role of cyclin D1 in supporting EBV infection in nasopharyngeal epithelial cells. While cyclin D1 is commonly expressed in nasopharyngeal carcinoma tissues we found that overexpression of cycline D1 is readily detected in dysplastic nasopharyngeal epithelial closely associated with EBV infection. Furthermore, using a panel of immortalized nasopharyngeal epithelial cells, we demonstrated that overexpression of cyclin D1 or CKD4 supports EBV infection and result in the establishment of stable EBV infected nasopharyngeal cell lines. We also observed that EBV infection induced growth inhibition and senescence in nasopharyngeal epithelial cells, which may be the underlying reason for low detection rate of EBV infection in healthy nasopharyngeal epithelial tissues. However, cyclin D1 overexpression suppressed senescence and differentiation in the immortalized nasopharyngeal epithelial cells, suggesting cyclin D1 may play an important role in supporting EBV infection in these cells. We postulated that the altered expression of cyclin D1 or CDK4 in premalignant nasopharyngeal epithelial cells may over-ride the growth suppression effects of EBV infection and support EBV latent infection in premalignant nasopharyngeal epithelial cells. Acknowledgement: This study is sponsored by grant support received from the Hong Kong Research Grant Council (Grant number: GRF780911 and AoE /M-06/08) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2037. doi:1538-7445.AM2012-2037

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells. © 2011 Hau et al., published_or_final_version

Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of 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-β 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 upon long term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage independent growth in soft-agar and exhibited invasive growth properties upon prolonged propagation. A distinguish 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 expression of MCP-1 and GRO-α. 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. Acknowlegement: The authors wish to acknowlege the funding support from the Research Grant Council (Hong Kong) and the Area of Excellence on Nasopharyangeal Carcinoma Research Center (Hong Kong). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2460.