Chi Man Tsang

The role of Epstein-Barr virus in epithelial malignancies.

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

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.

Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells

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 (CDK4R24C) 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.

Etiological factors of nasopharyngeal carcinoma

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.

Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: Regulation of infection and phenotypic characterization

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‐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 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‐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.

Cucurbitacin I elicits anoikis sensitization, inhibits cellular invasion and in vivo tumor formation ability of nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is an Asian-prevalent head and neck cancer with high invasiveness. Although several important risk factors for NPC development have been identified, there is currently no preventive strategy for NPC, even in endemic regions. Signal transducer and activator of transcription 3 (STAT3) has been implicated in NPC carcinogenesis, which may serve as a potential target for cancer prevention. Here, we examined the chemopreventive potential of Cucurbitacin I, a natural-occurring selective inhibitor of JAK/STAT3, in NPC models. We hypothesized that Cucurbitacin I would prevent NPC invasion and tumor formation. Our data demonstrated that brief exposure of NPC cells to Cucurbitacin I was sufficient to significantly reduce the in vitro clonogenicity and in vivo tumorigenicity of NPC cells. The chemopreventive potential of Cucurbitacin I was further demonstrated by pre-dosing of the animals with Cucurbitacin I prior to tumor inoculation, which was found to be able to suppress tumor growth up to 7 days post-inoculation. The anti-proliferation activity of Cucurbitacin I was accompanied by downregulation of phospho-STAT3 and STAT3 target gene expression (e.g. cyclin D1 and Mcl-1). Cucurbitacin I also reduced the invasiveness of invasive NPC cell lines with elevated STAT3 activation. Furthermore, our data demonstrated for the first time that Cucurbitacin I harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against human cancer. Taken together, our results suggested that Cucurbitacin I may be a potent chemopreventive agent for NPC with anti-invasion and anoikis-sensitizing activities.

STAT3 activation contributes directly to Epstein-Barr virus–mediated invasiveness of nasopharyngeal cancer cells in vitro†

Nasopharyngeal cancer (NPC) is an Epstein‐Barr virus (EBV)‐associated head and neck cancer prevalent in Asia. Although with reasons not fully understood, the intrinsic invasiveness of NPC is believed to be EBV‐linked. Recently, EBV was found to induce STAT3 activation. Constitutive STAT3 activation correlated with advanced clinical staging in NPC. We hypothesized that STAT3 activation by EBV directly contributes to the intrinsic invasiveness of NPC cells. Phospho‐STAT3‐Tyr705 was detected in high percentage of NPC tumors (7/10 cases). Using a paired NPC cell line model, HONE‐1 and the EBV‐infected counterpart, HONE‐1‐EBV, we found that HONE‐1‐EBV expressed a higher level of phospho‐STAT3‐Tyr705 and was ∼11‐fold more invasive than HONE‐1. In HONE‐1‐EBV, STAT3 siRNA targeting inhibited both spontaneous and serum‐induced invasion, as well as cell growth. Conversely, activation of STAT3 (by expressing an activated STAT3 mutant, namely STAT3C) in the parental HONE‐1, mimicking EBV‐induced STAT3 activation, significantly enhanced its invasiveness and proliferation, which was accompanied by increased expression of markers of mesenchymal status, proliferation and anti‐apoptosis. Our results demonstrated that EBV‐induced STAT3 activation is responsible for NPC cell proliferation and invasion. This was further confirmed by a small molecule inhibitor of JAK/STAT3, JSI‐124. JSI‐124 inhibited STAT3 activation in HONE‐1‐EBV, with subsequent growth inhibition, induction of PARP cleavage, abrogation of anchorage‐independent growth and invasion. We found that EBV‐independent activation of STAT3 by a growth factor, EGF, also contributed to NPC invasion. In conclusion, EBV‐induced STAT3 activation directly contributes to the intrinsic invasiveness of NPC cells and STAT3 targeting may be beneficial in treating aggressive NPC.

Enhanced IL-6/IL-6R Signaling Promotes Growth and Malignant Properties in EBV-Infected Premalignant and Cancerous Nasopharyngeal Epithelial Cells

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.

Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts.

BackgroundCortidis 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.MethodsIn 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.ResultsBerberine 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.ConclusionsOur 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.

Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein‐Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV‐positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV‐infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV‐positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP‐ribose) polymerase and caspase‐3, ‐7 and ‐9 in EBV‐positive versus EBV‐negative NPC cells were observed. More than 85% of NPC cells expressing immediate‐early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase‐3. Tracking of expression of EBV lytic proteins and cleaved caspase‐3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHAs induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus‐targeted therapy against EBV‐positive cancer.