Hong Bo Wang

Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells

Epstein–Barr virus (EBV) is implicated as an aetiological factor in B lymphomas and nasopharyngeal carcinoma. The mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells remain elusive. EBV glycoprotein B (gB) is the critical fusion protein for infection of both B and epithelial cells, and determines EBV susceptibility of non-B cells. Here we show that neuropilin 1 (NRP1) directly interacts with EBV gB23–431. Either knockdown of NRP1 or pretreatment of EBV with soluble NRP1 suppresses EBV infection. Upregulation of NRP1 by overexpression or EGF treatment enhances EBV infection. However, NRP2, the homologue of NRP1, impairs EBV infection. EBV enters nasopharyngeal epithelial cells through NRP1-facilitated internalization and fusion, and through macropinocytosis and lipid raft-dependent endocytosis. NRP1 partially mediates EBV-activated EGFR/RAS/ERK signalling, and NRP1-dependent receptor tyrosine kinase (RTK) signalling promotes EBV infection. Taken together, NRP1 is identified as an EBV entry factor that cooperatively activates RTK signalling, which subsequently promotes EBV infection in nasopharyngeal epithelial cells.

Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma

B‐lymphoma mouse Moloney leukemia virus insertion region 1 (Bmi1), a member of the polycomb group, has elevated expression and is involved in the pathogenesis of various aggressive cancers, including nasopharyngeal carcinoma (NPC). To date, the mechanisms underlying the high expression of Bmi1 in NPC remain obscure. To gain new insights into the transcriptional regulation of BMI1, we cloned and characterized the promoter region of BMI1. Luciferase reporter assays demonstrated that the region from −783 to +375 showed significant promoter activity. With the use of a series of 5′‐deletion and 3′‐deletion promoter constructs in luciferase reporter assays, the +167/+232 and −536/−134 regions were found to be sufficient for full promoter activity. Transcriptional activity of the BMI1 promoter was dependent on the Sp1 binding site cluster (+181/+214) as well as the E‐box elements (−181), and was abolished after mutation of the two cis‐elements. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that Sp1 bound to the region from +181 to +214 within the BMI1 promoter. In addition, gain‐of‐function and loss‐of‐function analyses revealed that Sp1 augmented Bmi1 expression. Further investigations using immunohistochemistry and quantitative RT‐PCR disclosed a significant positive correlation between the expression of Sp1 and Bmi1 in normal nasopharyngeal epithelial cells, NPC cells, and NPC tissue specimens. In addition, Myc, the known transcription factor for BMI1 in neuroblastomas, also activated the transcription of BMI1 through binding to the E‐box element (−181) within its promoter, and showed a positive correlation with the mRNA level of BMI1 in NPC. In conclusion, these findings provide valuable mechanistic insights into the role of Sp1 and c‐Myc in BMI1 transcription in NPC, and suggest that targeting of Sp1 or c‐Myc may be a potential therapeutic strategy for NPC.

Down-regulation of Sp1 suppresses cell proliferation, clonogenicity and the expressions of stem cell markers in nasopharyngeal carcinoma

BackgroundTranscription factor Sp1 is multifaceted, with the ability to function as an oncogene or a tumor suppressor, depending on the cellular context. We previously reported that Sp1 is required for the transcriptional activation of the key oncogenes in nasopharyngeal carcinoma (NPC), including B-lymphoma mouse Moloney leukemia virus insertion region 1 (Bmi1) and centromere protein H (CENPH), but the role of Sp1 and its underlying mechanisms in NPC remained largely unexplored. The objective of this study was to investigate the cellular function of Sp1 and to verify the clinical significance of Sp1 as a potential therapeutic target in NPC.MethodsThe levels of Sp1 in the normal primary nasopharyngeal epithelial cells (NPECs) and NPC cell lines were analyzed by Quantitative Real-time RT-PCR (qRT-PCR) and Western blot. The location and expression of Sp1 in the NPC tissues were detected by immunohistochemistry staining (IHC). The effect of Sp1 knockdown on the cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype in NPC cells were evaluated by MTT, flow cytometry, clonogenicity analysis and sphere formation assay.ResultsThe mRNA and protein levels of Sp1 were elevated in NPC cell lines than in the normal primary NPECs. Higher expression of Sp1 was found in NPC tissues with advanced clinical stage (P = 0.00036). Either inhibition of Sp1 activity by mithramycin A, the FDA-approved chemotherapeutic anticancer drug or Sp1 silencing by two distinct siRNA against Sp1 suppressed the growth of NPC cells. Mechanism analysis revealed that Sp1 silencing may suppress cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype through inducing the expression of p27 and p21, and impairing the expressions of the critical stem cell transcription factors (SCTFs), including Bmi1, c-Myc and KLF4 in NPC cells.ConclusionsSp1 was enriched in advanced NPC tissues and silencing of Sp1 significantly inhibited cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype of NPC cells, suggesting Sp1 may serve as an appealing drug target for NPC.

Nonmuscle myosin heavy chain IIA mediates Epstein–Barr virus infection of nasopharyngeal epithelial cells

Significance EBV causes nasopharyngeal carcinoma (NPC), an endemic disease in southern China. Unlike EBV infection of B lymphocytes, the molecular mechanisms and outcomes of EBV infection of epithelial cells are poorly understood. In vitro, EBV infects B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized nasopharyngeal epithelial cells (NPECs) poorly. We have now established a protocol to infect sphere-like cells derived from monolayer NPECs with EBV. Using this model system, we identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as an important host factor to mediate EBV infection of NPECs. NMHC-IIA knock-down substantially decreased NPEC EBV infection. This improved EBV infection model is likely to enable new insights into the mechanisms of EBV epithelia cell infection and will be useful for the understanding EBV’s role and effects in conversion of NPECs to NPC. EBV causes B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC). Although the mechanisms by which EBV infects B lymphocytes have been extensively studied, investigation of the mechanisms by which EBV infects nasopharyngeal epithelial cells (NPECs) has only recently been enabled by the successful growth of B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs in vitro and the discovery that neuropilin 1 expression positively affects EBV glycoprotein B (gB)-mediated infection and tyrosine kinase activations in enhancing EBV infection of BMI1-immortalized NPECs. We have now found that even though EBV infected NPECs grown as a monolayer at extremely low efficiency (<3%), close to 30% of NPECs grown as sphere-like cells (SLCs) were infected by EBV. We also identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as another NPEC protein important for efficient EBV infection. EBV gH/gL specifically interacted with NMHC-IIA both in vitro and in vivo. NMHC-IIA densely aggregated on the surface of NPEC SLCs and colocalized with EBV. EBV infection of NPEC SLCs was significantly reduced by NMHC-IIA siRNA knock-down. NMHC-IIA antisera also efficiently blocked EBV infection. These data indicate that NMHC-IIA is an important factor for EBV NPEC infection.

Anti-tumour activity of longikaurin A (LK-A), a novel natural diterpenoid, in nasopharyngeal carcinoma

BackgroundLongikaurin A is a natural ent-kaurene diterpenoid isolated from Isodon genus. The ent-kaurene diterpenoids isolated from medicinal plants have been shown to have anti-disease effects. The present study was designed to examine the anti-tumour effects of longikaurin A (LK-A) in nasopharyngeal carcinoma in vitro and in vivo.MethodsApoptosis and cell cycle arrest were determined by flow cytometry analysis of the cells treated with Longikaurin A. The proteins of apoptosis signaling pathway were detected by western blotting analysis. Finally, we examined whether LK-A exhibits anti-tumour activity in xenograft models.ResultsLongikaurin A inhibited the cell growth by inducing apoptosis and cell cycle arrest. At low concentrations, longikaurin A induced S phase arrest and at higher concentrations, longikaurin A induced caspase-dependent apoptosis by regulating apoptotic molecules. Finally, longikaurin A significantly inhibited the tumour growth of CNE2 xenografts in vivo and showed no obvious effect on the body weights of the mice.ConclusionOur results suggest that Longikaurin A exhibited anti-tumour activity in nasopharyngeal carcinoma in vitro and in vivo.

Proline-rich tyrosine kinase 2 and its phosphorylated form pY881 are novel prognostic markers for non-small-cell lung cancer progression and patients’ overall survival

Background:Our previous study revealed that proline-rich tyrosine kinase 2 (Pyk2) is implicated in both anchorage-independent growth and anoikis resistance in lung cancer cells. This study aims to explore the expression and clinical significance of Pyk2 and its phosphorylated forms in non-small-cell lung cancer (NSCLC).Methods:The mRNA and protein levels of Pyk2 or cancer stem cell markers (ALDH1a1, ABCG2 and Bmi-1) were either examined by reverse transcription–PCR or western blotting. An immunohistochemistry (IHC) assay was conducted to analyse the expression of Pyk2 and its phosphorylated forms in 128 NSCLC cases.Results:The levels of Pyk2 mRNA, total protein, and its phosphorylated form pY881 were higher in lung cancer lesions than in the paired noncancerous tissues. The IHC analysis showed the levels of the Pyk2 and Pyk2[pY881] proteins were highly expressed in 70 (54.7%) and 77 (60.2%) cases, respectively. Both Pyk2 and Pyk2[pY881] were independent prognostic factors for NSCLC patients. The gain and loss study of Pyk2 function revealed that Pyk2 could upregulate the expression of ALDH1a1, ABCG2 and Bmi-1 and enhance the ability of colony formation in soft agar assay in A549 and H460 cells.Conclusion:Both Pyk2 and phosphorylated Pyk2[pY881] are potential prognostic factors and therapeutic targets for NSCLC.

Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry.

Epstein–Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas1. EBV infects both B cells and epithelial cells2. Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells3. However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR–Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi’s sarcoma-associated herpesvirus infection through EphA24. Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.The receptor tyrosine kinase EphA2 is found to be an important determinant of EBV entry and fusion in epithelial cells. EphA2 depletion or absence, or the use of anti-EphA2 antibodies, inhibits oropharyngeal epithelial cell infection.

ISG15 predicts poor prognosis and promotes cancer stem cell phenotype in nasopharyngeal carcinoma.

Interferon-stimulated gene 15 (ISG15), the first identified ubiquitin-like protein, is known for its anti-viral capacity. However, its role in tumorigenesis remains controversial. Here, using RNA-seq profiling analysis, we identified ISG15 as a differentially expressed gene in nasopharyngeal carcinoma (NPC) and validated its overexpression in NPC samples and cells. High ISG15 levels in NPC tissues were correlated with more frequent local recurrence and shorter overall survival and disease-free survival. ISG15 overexpression promoted a cancer stem cell phenotype in NPC cells, including increased colony and tumorsphere formation abilities, pluripotency-associated genes expression, and in vivo tumorigenicity. By contrast, knockdown of ISG15 attenuated stemness characteristics in NPC cells. Furthermore, overexpression of ISG15 increased NPC cell resistance to radiation and cisplatin (DDP) treatment. Our study demonstrates a protumor role of ISG15, and suggests that ISG15 is a prognostic predictor and a potential therapeutic target for NPC.

Comprehensive profiling of EBV gene expression in nasopharyngeal carcinoma through paired-end transcriptome sequencing

The latent expression pattern of Epstein-Barr Virus (EBV) genes in nasopharyngeal carcinoma (NPC) has been extensively investigated, and the expression of several lytic genes in NPC has been reported. However, comprehensive information through EBV transcriptome analysis in NPC is limited. We performed paired-end RNA-seq to systematically and comprehensively characterize the expression of EBV genes in NPC tissue and C666-1 NPC cell line, which consistently carries EBV. In addition to the transcripts restricted to type II latency infection, the type III latency EBNA3s genes and a substantial number of lytic genes, such as BZLF1, BRLF1, and BMRF1, were detected through RNA-seq and were further verified in C666-1 cells and NPC tissue through realtime PCR.We also performed clustering analysis to classify NPC patient groups in terms of EBV gene expression, which presented two subtypes of NPC samples. Results revealed interesting patterns of EBV gene expression in NPC patients. This clustering was correlated with many signaling pathways, such as those related to heterotrimeric G-protein signaling, inflammation mediated by chemokine and cytokine signaling, ribosomes, protein metabolism, influenza infection, and ECM-receptor interaction. Our combined findings suggested that the expression of EBV genes in NPC is restricted not only to type II latency genes but also to type III latency and lytic genes. This study provided further insights into the potential role of EBV in the development of NPC.

Lactate Dehydrogenase Is an Important Prognostic Indicator for Hepatocellular Carcinoma after Partial Hepatectomy

Preoperative serum lactate dehydrogenase (LDH) has been used as a prognostic indicator for patients with hepatocellular carcinoma (HCC) treated with sorafenib or undergoing transcatheter arterial chemoembolization, but its significance in predicting survival of HCC patients who received curative resection remains undefined. A total of 683 patients with histopathologically confirmed HCC were enrolled in this study. The prognostic significance of preoperative serum LDH was determined by Kaplan-Meier analysis and a Cox proportional hazards regression model. The association between the preoperative serum LDH and clinicopathological parameters was evaluated by the χ2 test or linear regression analysis when appropriate. Higher preoperative serum LDH level was associated with worse prognosis. In a multivariate Cox proportional hazards analysis, the preoperative serum LDH level could predict overall survival and recurrence independently. Higher preoperative serum LDH level is associated with the elevated serum alpha-fetoprotein, the presence of hepatitis B surface antigen, larger tumor size, the presence of macrovascular invasion, the advanced tumor–lymph node–metastasis stage, worse tumor differentiation, and Child-Pugh B. Preoperative serum LDH level was an inexpensive, simple, convenient, and routinely measured biomarker exhibiting a potential to select patients at high risk with poor clinical outcome for appropriate treatment strategies.