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In our previous whole-transcriptome sequencing analysis, downregulation of a long non-coding RNA, maternally expressed gene 3 (MEG3), was identified in NPC samples. This finding suggests the possible role of MEG3 as a tumor suppressor in this distinctive disease. In the present study, two MEG3 variants, AF119863 (MEG3-AF) and BX247998 (MEG3-BX), were found abundantly expressed in a normal nasopharyngeal epithelial cell line, NP69. Significant downregulation of MEG3-AF was further verified in a panel of NPC samples including xenografts and primary biopsies. MEG3 is an imprinted gene located within chromosome 14q32, a common deleted region in NPC. Both DNA copy number loss and aberrant promoter methylation contributed to MEG3 inactivation. Interestingly, MEG3 expression could successfully be rescued by the treatment of a demethylation agent. Besides, ectopic expression of MEG3 in NPC cell lines resulted in considerable repression of in vitro anchorage-independent growth and in vivo tumorigenicity, in addition to significant inhibition in cell proliferation, colony formation, and induction of cell cycle arrest. Finally, we revealed the association between MEG3 activity and the p53 signaling cascade. Our findings characterize MEG3 as a tumor suppressive long non-coding RNA in NPC and encourage the development of precise long non-coding RNA-targeted epigenetic therapy against this malignancy. © 2016 Wiley Periodicals, Inc.

Background Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy that exhibits distinct geographical and ethnic prevalence. Although the contemporary therapeutic approach of radio-/chemotherapy provides excellent results for patients with early-stage disease, it is far from satisfactory for those with disease remission and distant metastasis. Promising therapeutic strategies for advanced and relapsed NPC are still lacking. We recently identified and characterized a cancer stem-like cell (CSC) subpopulation in NPC that appeared to play an important role in tumor progression. Microarray analysis revealed downregulation of several stemness-inhibiting miRNAs in these CSC cells. Among these miRNAs, miR-96 and miR-183 showed the highest fold change and were selected to elucidate their role in repressing NPC CSC properties. Methods MiR-96 and miR-183 expression in NPC CSCs was detected by qRT-PCR. Transient and stable transfection was performed in EBV-positive NPC C666-1 cells to examine the effects of ectopic expression of miR-96 and miR-183 on repressing cell growth and CSC properties. Anchorage-dependent (colony formation) and anchorage-independent (tumor sphere formation) growths of these miR-96 and miR-183 expressing cells were determined. Expression of multiple CSC markers and related molecules were accessed by flow cytometry and Western blotting. The tumorigenicity of the stable miR-96- and miR-183-transfected NPC cells was examined in an in vivo nude mice model. Results Downregulation of miR-96 and miR-183 was confirmed in NPC spheroids. Using transient or stable transfection, we showed that ectopic expression of miR-96 and miR-183 suppressed cell growth and tumor sphere formation in NPC. Reduced NICD3 and NICD4 in miR-96- and miR-183-expressing NPC cells suggests the involvement of the NOTCH signaling pathway in their tumor suppressive function. Finally, we showed that the tumorigenicity of cells stably expressing miR-183 was significantly inhibited in the in vivo nude mice model. Conclusions miR-183 is a tumor-suppressive miRNA in EBV-associated NPC. Its abilities to suppress CSC properties in vitro and effectively reduce tumor growth in vivo shed light on its role as a potential therapeutic target. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2525-5) contains supplementary material, which is available to authorized users.

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

In our previous whole-transcriptome sequencing analysis, downregulation of a long non-coding RNA, maternally expressed gene 3 (MEG3), was identified in NPC samples. This finding suggests the possible role of MEG3 as a tumor suppressor in this distinctive disease. In the present study, two MEG3 variants, AF119863 (MEG3-AF) and BX247998 (MEG3-BX), were found abundantly expressed in a normal nasopharyngeal epithelial cell line, NP69. Significant downregulation of MEG3-AF was further verified in a panel of NPC samples including xenografts and primary biopsies. MEG3 is an imprinted gene located within chromosome 14q32, a common deleted region in NPC. Both DNA copy number loss and aberrant promoter methylation contributed to MEG3 inactivation. Interestingly, MEG3 expression could successfully be rescued by the treatment of a demethylation agent. Besides, ectopic expression of MEG3 in NPC cell lines resulted in considerable repression of in vitro anchorage-independent growth and in vivo tumorigenicity, in addition to significant inhibition in cell proliferation, colony formation, and induction of cell cycle arrest. Finally, we revealed the association between MEG3 activity and the p53 signaling cascade. Our findings characterize MEG3 as a tumor suppressive long non-coding RNA in NPC and encourage the development of precise long non-coding RNA-targeted epigenetic therapy against this malignancy. © 2016 Wiley Periodicals, Inc.

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which is prevalent in south-east Asia and southern China. Despite the multiple genetic and epigenetic changes reported, the contribution of dysregulated signalling pathways to this distinct type of head and neck cancer is not well understood. Here we demonstrate the up-regulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) in the majority of EBV-positive tumour lines and primary tumours. Among the NOTCH receptors, NOTCH3 was over-expressed in all EBV-positive tumour lines and 92.5% of primary tumours. Aberrant activation of NOTCH3 signalling was consistently detected in all these samples. These findings imply that NOTCH3 may play an crucial role in the development of NPC. By NOTCH3 specific siRNA, NOTCH3 signalling was suppressed and thereby significant growth inhibition and apoptosis induction occurred in NPC cells. Down-regulation of a number of targets involved in cell proliferation, eg CCND1, C-MYC,NFKB1, and survival, eg BCL2, BCL-XL, SURVIVIN, was confirmed in the NOTCH3 knockdown NPC cells. Importantly, NOTCH3 knockdown highly enhanced the sensitivity of NPC cells to cisplatin treatment. Furthermore, we revealed that the ability of NPC cells to form spheroids in vitro and tumours in nude mice was also significantly decreased after knockdown of NICD3 expression. These findings indicate that activation of NOTCH3 pathway is a critical oncogenic event in NPC tumourigenesis. Targeting NOTCH3 signalling may serve as a potential therapeutic approach for treating patients suffering from EBV-associated NPC.

Viral loads and cytokine responses Epstein–Barr virus (EBV) were measured in an 18-year-old boy with severe glandular fever complicated by a mild anaemia, severe thrombocytopaenia and neutropaenia. Hepatosplenomegaly was detected by abdominal ultrasound in the presence of significant hepatitis. Cytokine testing demonstrated elevated cell-mediated Th1 (IFN-γ, IL-12, sTNFR1, CXCL10, CXCL9 and CCL3) and humoral Th2 (IL-4) immune responses. Serum antibodies to EBV virus capsid antigen (VCA) IgM and IgG antibodies were detected, together with a raised EBV DNA level (up to about 70,000 DNA copies/mL) in the acute phase of the illness. This EBV DNA load decreased rapidly in response to treatment with a combination of foscarnet, intravenous immunoglobulin and prednisolone, and the boy's symptoms settled eventually after approximately 50 days of illness, following this combined antiviral and immune-modulating therapy. Detailed immunological, virological, haematological and biochemical laboratory parameters are presented to document this patient's severe EBV disease and eventual recovery. J. Med. Virol. 81:99–105, 2009. © 2008 Wiley-Liss, Inc.

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

Monoclonal antibody against TNF-alpha such as infliximab has shown clinical efficacy in controlling the inflammatory signs and symptoms of rheumatoid arthritis (RA), but the detailed immunotherapeutic mechanism is not fully understood. We investigated 19 patients with active RA who were treated with infliximab (3 mg/kg) at weeks 0, 2, 6 and 14. Peripheral blood was obtained from the patients at weeks 0 and 14 and cultured with mitogens phytohaemagglutinin (PHA) and lipopolysaccharide (LPS). The concentrations of cytokines and soluble adhesion molecules (sICAM-1, sICAM-3, sE-selectin, sP-selectin, sVCAM-1 and sPECAM-1) in supernatant fluids or plasma were measured by flow cytometry and ELISA. After infliximab treatment, the absolute and percentage increases in release of inflammatory cytokine TNF-alpha and potent neutrophil chemoattractant IL-8 upon PHA and LPS activation were significantly decreased when compared to those of before treatment (all P0.01). The increased releases of IL-6, IL-1beta, IL-18 and IL-12 upon mitogen activation were similar before and after infliximab treatment (all P0.05). Plasma concentrations of these cytokines and soluble adhesion molecules did not differ significantly before and after infliximab treatment. Our study suggests that the reduction in synovial inflammation may be due to the decreased production of TNF-alpha and IL-8, and hence the number of neutrophils and other pro-inflammatory leukocytes infiltrating into the inflamed sites.

SummaryT-bet is a novel transcription factor regulating lineage commitment of T helper (Th) lymphocytes to a predominant Th1 phenotype. Previous studies on T-bet and asthma focused mainly on bronchial biopsy specimens. This study assessed the relationship between T-bet expression and levels of selected chemokines in the peripheral blood of asthmatics. Blood was collected from 24 steroid-naive asthmatics, 39 asthmatics on inhaled corticosteroid and 32 age- and sex-matched controls for assay of T-bet expression, specific IgE and chemokines (interferon-gamma inducible protein-10 (IP-10/CXCL10), monokines induced by interferon-gamma (MIG/CXCL9), monocyte chemotactic protein-1 (MCP-1/CCL2), regulated upon activation normal T cell expressed and secreted (RANTES/CCL5) and interleukin-8 (IL-8/CXCL8) levels. T-bet mRNA expression was assessed by real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR). Chemokine levels were assessed by immunofluorescence flow cytometry. The mean (s.d.) age and forced expiratory volume in 1 s (FEV1)% predicted of the asthmatics were 43·6 (14·6) years and 85·9 (20·0)%, respectively. The median (IQR) T-bet expression after normalization with β-actin was suppressed in asthmatics versus controls [asthmatics 0·71 (0·59) versus controls 1·07 (1·14), P = 0·03]. The median (IQR) of plasma RANTES was elevated, whereas IP-10 was suppressed in asthmatics versus controls (RANTES: 13 658·0 (13673·3) versus 6299·5 (19 407·8) pg/ml, P = 0·03; IP-10: 1047·6 (589·8) versus 1306·4 (759·9) pg/ml, P = 0·001). There was a weak and negative correlation between T-bet expression and RANTES level in the asthmatics (r = –0·29, P = 0·032). T-bet could be measured in peripheral blood and its expression was suppressed in asthmatics. This is in keeping with asthma being a predominantly Th2 disease and T-bet probably plays a role in the pathogenesis of asthma. Further studies are needed to explore the potential application of peripheral blood monitoring of T-bet.

Although the Epstein-Barr virus (EBV) has spread to all populations in the world, EBV-associated nasopharyngeal carcinoma (NPC) is prevalent only in South China and Southeast Asia. The role of EBV in the malignant transformation of nasopharyngeal epithelium is the main focus of current researches. Radiotherapy and chemoradiotherapy have been successful in treating early stage NPC, but the recurrence rates remain high. Unfortunately, local relapse and metastasis are commonly unresponsive to conventional treatments. These recurrent and metastatic lesions are believed to arise from residual or surviving cells that have the properties of cancer stem cells. These cancer stem-like cells (CSCs) have the ability to self-renew, differentiate, and sustain propagation. They are also chemo-resistant and can form spheres in anchorage-independent environments. This review summarizes recent researches on the CSCs in EBV-associated NPC, including the findings regarding cell surface markers, stem cell-related transcription factors, and various signaling pathways. In particular, the review focuses on the roles of EBV latent genes [latent membrane protein 1 (LMP1) and latent membrane protein 2A (LMP2A)], cellular microRNAs, and adenosine triphosphate (ATP)-binding cassette chemodrug transporters in contributing to the properties of CSCs, including the epithelial-mesenchymal transition, stem-like transition, and chemo-resistance. Novel therapeutics that enhance the efficacy of radiotherapy and chemoradiotherapy and inhibitors that suppress the properties of CSCs are also discussed.

Nasopharyngeal carcinoma (NPC) is a common viral-associated neoplasm in which multiple signaling cascades are interfered with by Epstein-Bar virus (EBV) latent proteins and various genetic alterations. Aside from the previously reported PIK3CA amplification, we examined the role of INPP4B, a negative regulator of the PI3K/AKT signaling pathway in the development of NPC. By RT-PCR and Western blotting, we revealed that the expression of INPP4B was down-regulated in all five established EBV-positive tumor lines. While INPP4B was consistently expressed in normal nasopharyngeal epithelial cells, downregulation of INPP4B was found in 32/65 (49.2%) of primary tumors by immunohistochemistry. Furthermore, our study also demonstrated the hypermethylation of the 5′CpG island of INPP4B in the tumors in which INPP4B transcription was downregulated. Notably, the re-expression of INPP4B was detected in the NPC cells treated with the demethylation agent (5-aza-2′deoxycytidine). Our study showed that promoter hypermethylation was the major mechanism for transcriptional silencing of INPP4B in NPC. Furthermore, restoration of INPP4B expression significantly suppressed PI3K/AKT downstream signals in the NPC C666-1 cells. In vivo growth inhibition was clearly demonstrated in the tumor cells stably expressing INPP4B. The findings indicate that epigenetic inactivation of INPP4B is one of the key mechanisms in activating PI3K/AKT signaling cascade and playing a role in the tumorigenesis of NPC.

Nasopharyngeal carcinoma (NPC) is a distinctive type of squamous cell carcinoma of the head and neck region with remarkable ethnic and geographic distribution. It is consistently associated with Epstein–Barr virus (EBV) infection and serves as a fascinating model to understand the complex interaction among environmental, viral, and genetic factors in human tumourigenesis. The development of NPC involves clonal expansion of EBV-infected cells and cumulative genetic and epigenetic changes. Both viral and cellular genes contribute to disease pathogenesis and their interplay creates distinct phenotypes, unique molecular features and tumour microenvironment. Recent findings on EBV-encoded and cellular miRNAs demonstrate their importance in NPC progression by altering multiple cellular pathways. On the basis of current knowledge, we proposed a stepwise model describing the initiation events and the role of EBV latent infection and multiple genetic changes in NPC tumourigenesis. Key Concepts: Nonkeratinising NPC is consistently associated with EBV infection. The development of NPC involves EBV latent infection and cumulative genetic and epigenetic changes influenced by predisposing genetic factors and environmental carcinogens. Inactivation of tumour suppressor genes at chromosomes 3p and 9p and clonal expansion of EBV-infected cells are critical initiation events in NPC tumourigenesis. EBV-encoded and cellular miRNAs contribute to the NPC progression by altering multiple cellular pathways and modulating EBV latent gene expression. The interplay between viral and cellular genes contributes to the establishment of distinct phenotypes, unique molecular features and tumour microenvironment of NPC. Keywords: nasopharyngeal carcinoma; Epstein–Barr virus; tumour suppressor gene; oncogene; DNA methylation; miRNA; multistep tumourigenesis

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer arising from the epithelium of nasopharynx. While the incidence rate of NPC is low in Western countries, it is prevalent in southern China, south-east Asia and northern Africa. Generally, it is believed that multiple factors, including genetic predisposition, environmental carcinogens and Epstein-Barr virus infection, contribute to the tumor initiation and progression of NPC. Similar to other solid tumors in which genetic rearrangements play causal role in the genesis of tumors, our earlier cytogenetic and spectral karyotyping have demonstrated the prevalence of chromosomal translocations in NPC tumors. These evidences suggest that novel fusion gene product arises from chromosomal translocation may contribute to the pathogenesis of the disease. Using paired-end whole-transcriptome sequencing, we discovered a novel fusion transcript composed of the exon 1 of the ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and exon 7-9 of zinc finger protein 423 (ZNF423) on 16q12.1 from the NPC cell line C666-1. Moreover, FISH analysis using break-apart probes validated the UBR5-ZNF423 fusion. Furthermore reverse transcription-PCR (RT-PCR) confirmed the expression of the corresponding fusion transcript. Intriguingly, the fusion transcript was recurrently detected in 12/144 (8.3%) of primary tumors by RT-PCR, which indicates the UBR5-ZNF423 fusion gene may contribute to the genesis of a subset of NPCs. The growth of C666-1 cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by RNA interference inhibited the cell proliferation of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 mouse fibroblasts by soft agar colony formation assay. Moreover, NIH3T3 cells stably expressing the fusion protein induced tumor formation in nude mice. Furthermore, using an inducible shRNA (Lac operon-driven) targeting the fusion transcript, the growth of C666-1 tumor was suppressed in nude mice. These findings suggest that expression of UBR5-ZNF423 fusion protein contributes to the transformation of a subset of NPCs. Currently, we are working on the functional role(s) of the fusion protein in C666-1 cells. Preliminary results demonstrated that the fusion protein interacts with the Early B-cell Factor 3 (EBF3) and may deregulate EBF3 transcriptional activity. Since EBF3 is a tumor suppressor gene, deregulating EBF3 downstream gene expression by UBR5-ZNF423 may confer to its oncogenic function on NPCs. In conclusion, we have successfully identified the UBR5-ZNF423 fusion transcript in a subset of NPC tumors. By understanding the molecular mechanisms of this fusion protein on NPCs, novel therapeutic interventions may be implemented on treating a subsets of NPC tumors expressing this oncogenic fusion protein. Citation Format: Pok Man Hau, Grace Chung, Raymond Lung, Samantha Lun, Chit Chow, Alice Wong, Fei-Fei Liu, George Tsao, Kevin Yip, Ka Fai To, Kwok Wai Lo. The identification of UBR5-ZNF423 recurrent fusion gene in EBV-associated nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4064.

Background: Metastatic colorectal cancer (mCRC) is a heterogeneous disease with a poor prognosis. By using high resolution array comparative genomic hybridization (aCGH) and mRNA gene expression microarray of mCRC samples, we found up-regulation of RASAL2, a RAS GAP gene that is located on chromosome 1q, in colorectal tumors. Oncogenic RAS represents one of the most common molecular changes in human colorectal adenocarcinoma. However, the role of RASAL2 in colorectal adenocarcinoma metastasis and the related mechanisms are still unclear. We analyzed its aberrant expression, clinical significance and biological effects in colorectal cancer. Methods: Expression of RASAL2 was examined by QRT-PCR, western blot and immunohistochemistry in CRC cell lines, primary and metastatic CRC and the corresponding normal colonic mucosa. The biological effects of RASAL2 on proliferation, apoptosis, cell cycle, and cell motility and invasiveness were evaluated by siRNA knock down and RASAL2 reconstitution in CRC cell lines. Results: Up-regulation of RASAL2 mRNA was observed in CRC cell lines (n = 9) than normal colon mucosal tissues. Interestingly, significantly higher RASAL2 mRNA was found in cell lines derived from advanced stage tumors (n = 4, Dukes’ C and D) than those from early stage tumors (n = 5, Dukes’ A and B) (P = 0.0317). Up-regulation of RASAL2 mRNA was also found in primary CRCs (n = 77) compared with normal colon mucosa (n = 18, P We knocked down RASAL2 by siRNA in 2 CRC cell lines (DLD1 and HCT116) with high endogenous RASALs level. Successful knockdown was demonstrated by western blot analysis. SiRASAL2 significantly inhibited cell proliferation (P Conclusions: Up-regulation of RASAL2 was frequently found in CRC cell lines and primary and metastatic tumors. Our experimental findings suggested that RASAL2 might play an oncogenetic role in CRC and promotes tumor invasion and metastasis. A better understanding of the molecular mechanism of RASAL2 in promoting CRC cell metastasis may lead to a more effective management of mCRC patients. Citation Format: Yi PAN, Joanna Hung Man TONG, Raymond Wai Ming LUNG, Samantha Wei Man LUN, Kwok Wai LO, Anthony Wing Hung CHAN, Ka Fai TO. RASAL2 promotes tumor progression and metastasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1529.

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.

Background Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis. Methods Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed. Results 63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2–15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2–27 times lower than seasonal influenza; P−values

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which is prevalent in south-east Asia and southern China. Despite the multiple genetic and epigenetic changes reported, the contribution of dysregulated signalling pathways to this distinct type of head and neck cancer is not well understood. Here we demonstrate the up-regulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) in the majority of EBV-positive tumour lines and primary tumours. Among the NOTCH receptors, NOTCH3 was over-expressed in all EBV-positive tumour lines and 92.5% of primary tumours. Aberrant activation of NOTCH3 signalling was consistently detected in all these samples. These findings imply that NOTCH3 may play an crucial role in the development of NPC. By NOTCH3 specific siRNA, NOTCH3 signalling was suppressed and thereby significant growth inhibition and apoptosis induction occurred in NPC cells. Down-regulation of a number of targets involved in cell proliferation, eg CCND1, C-MYC,NFKB1, and survival, eg BCL2, BCL-XL, SURVIVIN, was confirmed in the NOTCH3 knockdown NPC cells. Importantly, NOTCH3 knockdown highly enhanced the sensitivity of NPC cells to cisplatin treatment. Furthermore, we revealed that the ability of NPC cells to form spheroids in vitro and tumours in nude mice was also significantly decreased after knockdown of NICD3 expression. These findings indicate that activation of NOTCH3 pathway is a critical oncogenic event in NPC tumourigenesis. Targeting NOTCH3 signalling may serve as a potential therapeutic approach for treating patients suffering from EBV-associated NPC.

A recently identified interleukin (IL)-17-producing T-helper (Th) lymphocyte subset, which comprises Th17 cells producing hallmark cytokines IL-17A, IL-17F and IL-22, is involved in chronic inflammatory diseases. Elevated gene and protein expressions of IL-17 are manifested in allergic asthma. We further characterized the activation of Th17 cells in asthmatic patients. Peripheral blood mononuclear cells (PBMC) were purified from 31 asthmatic patients and 20 sex- and age-matched control subjects. The number of IL-17A secreting cells in peripheral blood was enumerated by enzyme-linked immunosorbent spot assay. Cell surface expression of Th17-related chemokine receptor CCR6, and plasma level of IL-17A, IL-17F and IL-22, and ex vivo production of IL-17A and IL-22 were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. The number of peripheral Th17 lymphocytes, expression of CCR6 on Th cells, and ex vivo IL-23, anti-CD3 and anti-CD28 induced production of IL-22 by PBMC were significantly elevated in asthmatic patients compared with control subjects (all p0.01). This clinical study further confirmed increased number of peripheral Th17 lymphocytes and cell surface expression of CCR6 receptors on Th cells in asthmatic patients. Pro-inflammatory cytokine IL-23 can exacerbate disease severity by activating pathogenic Th17 lymphocytes to release downstream inflammatory cytokine IL-22 in asthma.

We investigated the expression profile of toll-like receptors (TLRs) and TLR ligand-activated production profile of asthma-related inflammatory cytokines in asthmatic patients. The expression of TLR1–8 on monocytes, CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, CD19+ B lymphocytes, and dendritic cells, and ex vivo production of cytokines from peripheral blood mononuclear cells activated by TLR ligands were measured by flow cytometry. Ex vivo productions of TNF-α, IL-10, and IL-1β by TLR4 and TLR5 ligand LPS and flagellin were significantly lower in asthmatic patients (all P

Allergic diseases such as asthma and allergic dermatitis are associated with the degranulation of mast cells. Chymase, a mast-cell-specific protease, is the major component in mast cell granules that can induce eosinophil infiltration into inflammatory sites. We examined the immunopathological mechanisms for the activation of eosinophils by chymase in allergic inflammation. Cytokines were measured by cytometric bead array Flex Sets multiplex assay using flow cytometry and enzyme-linked immunosorbent assay. Adhesion molecules, migration and intracellular signalling pathways were assessed by flow cytometry, Boyden chamber assay and Western blot, respectively. Chymase suppressed the apoptosis of eosinophils and induce the release of the cytokine interleukin-6 (IL-6) and chemokines CXCL8, CCL2 and CXCL1 by eosinophils dose-dependently. It also up-regulated the surface expression of adhesion molecule CD18 and stimulated the chemokinetic migration of eosinophils. The expressions of adhesion molecules, cytokines and chemokines, and chemokinetic migration were differentially regulated by the activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Akt, Janus-activated kinase and nuclear factor-kappaB pathways. Chymase therefore plays a pivotal immunological role in the interaction between mast cells and eosinophils in allergic diseases such as allergic dermatitis by inducing adhesion molecule-mediated chemokinetic migration and inflammatory cytokines and chemokines of eosinophils, through multiple intracellular signalling molecules and transcription factor. Our results therefore provide a further biochemical basis for the pathogenesis of allergic inflammation consequent on the interaction between mast cells and eosinophils, and give insight for the development of new therapies.

This study further elucidates the roles of selected chemokines (IP-10, MIG, and RANTES) and their receptors (CCR3, CCR5, and CXCR3) in asthma. We compared their profiles in six groups of participants—atopic cohort and nonatopic cohort (each including controls and asthmatic patients with or without steroid therapy). Plasma concentration of IP-10 was significantly lower while that of RANTES and the expression of CCR3 were higher in asthmatic patients (all p < 0.05). Plasma RANTES correlated positively with the GINA severity score in all asthmatic patients (r=0.27, p < 0.05), and with IL-13 in nonatopic asthmatic patients (r=0.46, p < 0.05). In asthmatic patients, the ex vivo release of IP-10 and MIG was attenuated in PBMC activated with allergen, mitogens and IL-18 (p < 0.05). In conclusion, plasma RANTES may be a surrogate marker for asthma and the diminished Th1 related CXC chemokine production may contribute to Th2 predominance in asthma.

SummaryThe co-stimulatory interactions of the B7 family molecules CD80 and CD86 on antigen-presenting cells, together with their T cell counter receptors CD28 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), modulate T lymphocyte-mediated immune responses in a reciprocal manner. To investigate whether there is altered expression and the clinical significance of soluble co-stimulatory molecules in asthmatic patients, plasma concentrations of sCTLA-4, sCD28, sCD80 and sCD86 in 51 adult allergic asthmatic adults with or without steroid treatment, and 35 sex- and age-matched control subjects were measured by enzyme-linked immunosorbent assay (ELISA). Cell surface expression of CTLA-4 and CD28 on peripheral blood mononuclear cells (PBMC) were analysed by flow cytometry. Results showed that the plasma sCTLA-4 concentration was significantly higher in all asthmatic patients while sCD28 and sCD86 concentrations were significantly higher in steroid and non-steroid treated asthmatic patients, respectively, compared with control subjects (all P < 0·01). Significantly increased cell surface expression of CD28 but not CTLA-4 on PBMC was found in asthmatic patients compared with controls (P < 0·05). The plasma concentration and cell surface expression of CTLA-4 were found to exhibit positive and significant correlations with those of CD28 (both P < 0·05). Serum total IgE concentration correlated positively and significantly with sCTLA-4 and sCD28 concentrations in allergic asthmatic patients (both P < 0·05). The increased expression of these soluble co-stimulatory molecules may reflect the dysregulation of T cell activation, thereby contributing to the immunopathogenesis of allergic asthma.

BACKGROUND: As a distinctive type of head and neck cancers, nasopharyngeal carcinoma (NPC) is genesis from the clonal Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cells accumulated with multiple genetic lesions. Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC. In addition to the reported CDKN2A/p16, herein, we elucidated the role of a miRNA, miR-31 within this 9p21.3 region as NPC-associated tumor suppressor. METHODS: The expression and promoter methylation of miR-31 were assessed in a panel of NPC tumor lines and primary tumors. Its in vitro and in vivo tumor suppression function was investigated through the ectopic expression of miR-31 in NPC cells. We also determined the miR-31 targeted genes and its involvement in the growth in NPC. RESULTS: Downregulation of miR-31 expression was detected in almost all NPC cell line, patient-derived xenografts (PDXs) and primary tumors. Both homozygous deletion and promoter hypermethylation were shown to be major mechanisms for miR-31 silencing in this cancer. Strikingly, loss of miR-31 was also obviously observed in the dysplastic lesions of nasopharynx. Restoration of miR-31 in C666-1 cells inhibited the cell proliferation, colony-forming and migratory capacities. Dramatic reduction of in vitro anchorage-independent growth and in vivo tumorigenic potential were demonstrated in the stable clones expressing miR-31. Furthermore, we proved that miR-31 suppressed the NPC cell growth via targeting FIH1 and MCM2. CONCLUSIONS: The findings provide strong evidence to support miR-31 as a new NPC-associated tumor suppressor on 9p21.3 region. The inactivation of miR-31 may contribute to the early development of NPC., published_or_final_version

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which is prevalent in southern China and south-east Asia. EBV-encoded EBNA1 is consistently expressed in NPC cells and implicates the replication and stable persistence of latent viral genome. As a phosphorylated protein, the function of EBV is believed to be affected by phosphorylation.In this study, we aimed to investigate whether the phospho-Ser/Thr-Pro specific prolyl-isomerase PIN1 alters EBNA1 function and plays a role in the tumorigenesis of EBV-associated NPC.By western blotting and immunohistochemical staining, we have found that PIN1 was highly expressed in almost all 6 NPC tumor lines and 70 primary tumors. In the EBV-positive C666-1 cells, co-localization and interaction of EBNA1 and PIN1 was detected by immunofluorence staining and co-immunoprecipitation assay respectively. The interaction between PIN1 and EBNA1 at the specific serine-proline motif (Ser383 and Ser393 in EBNA1;WW domain in Pin1) was also demonstrated. To investigate potential role of PIN1 in NPC tumorigenesis, we established a stable nasopharyngeal epitheliacell line NP69 overexpressing PIN1. In this model, we found that overexpression of PIN1 up-regulated cyclin D1 and activated MAPK/JNK pathway. Increased anchorage-independent growth of NP69 overexpressing PIN was shown in soft agar assay.To further validate the oncogencity of PIN1, we knocked down its expression in the NPC cell line C666-1 by RNA interference (siRNA). The study confirmed the PIN1-mediated cyclin D1 suppression. Importantly, knocking down of PIN1 significantly inhibited the cell proliferation, DNA synthesis and migration of NPC cells.Similar findings were also detected in the NPC cells treated with PIN1 inhibitor, Juglone. Treating EBV-positive C666-1cell with Juglone,we found it can significantly induce apoptosisand suppress the cyclinD1expression in C666-1cells. The anti-tumor effect of PIN inhibitor was demonstrated in the in vitro NPC model. To elucidate the anti-tumor potential of PIN1 inhibitorin vivo, the mice implanted with NPC cells were subjected to treatment with different doses of Juglone. A dose-dependent inhibition of tumor growth was observed in the mice treated with PIN1 inhibitor. In conclusion, our findings imply that PIN1overexpression plays important role in the development of NPC. Targeting Pin1 may serve as a potential therapeutic approach for treating patients suffering from this EBV-associated cancer. Citation Format: Meng Xu, Chit Chow, Chartia Ching-Mei Cheung, Chi-Man Tsang, Samantha Wei-Man Lun, Jessie Wai-Fung Yuen, Grace Tin-Yun Chung, Sah-Wah Tsao, Ka Fai To, Kwok Wai Lo. Inhibition of PIN1 suppresses tumorigenicity of EBV-associated NPC. [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 2189. doi:10.1158/1538-7445.AM2013-2189

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignant cancer prevalent in Southern China and South-eastern Asia. In this study, we aim to elucidate the fundamental role of aberrant expressed microRNAs (miRNA) in NPC tumorigenesis. By comparison of miRNA expression profiles in NPC tumor lines with non-malignant nasopharyngeal epithelial cell, 119 differentially expressed miRNAs were identified in EBV-associated NPC. Among the 58 microRNAs downregulated in NPC, transcriptional silencing of miR-31 was consistently found in both NPC tumor lines and primary tumors. Expression of miR-31 was detected in none of 6 EBV-positive tumor lines and 38 microdissected primary tumors, but in all normal nasopharyngeal epithelia. Interestingly, miR-31 is located at 0.5 Mb telomeric to CDKN2A at 9p21.3, which is commonly homozygous deleted in NPC. Homozygous deletion of both miR-31 and CDKN2A locus was confirmed in 2 tumor lines. In the 4 tumor lines with intact miR-31, hypermethylation of 5′CpG islands was detected by bisulfate sequencing and MSP analysis. Re-expression of miR-31 was demonstrated in the EBV-positive NPC cell line C666-1 treated with 5-aza-2′-deoxycytidine. The findings suggested that homozygous deletion and promoter methylation are the major mechanisms for transcriptional silencing of miR-31 in NPC. By microarray and bioinformatic analysis, we have identified a number of putative targets of miR-31. Among these candidates, we have proven that miR-31 targeted FIH1 and MCM2 expression in NPC. Ectopic expression of miR-31 in NPC cells resulted in repression of FIH1 and MCM2 protein. Finally, we have demonstrated that restoration of miR-31 or knockdown of FIH1 expression significantly suppressed cell proliferation of C666-1. Our findings indicated that miR-31 is a NPC-associated microRNA which negatively regulates cell growth by repressing FIH1 expression. 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 2288. doi:1538-7445.AM2012-2288

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which prevalent in Southeast Asia and Southern China. In an effort to seek for new targets for therapies, we have elucidated the underlying mechanisms for dysregulated signaling pathways in this EBV-associated cancer. Our recent study showed that constitutive activation of NOTCH pathway is consistently detected in both NPC tumor lines and primary tumors. Expression of EBV latent genes also altered the NOTCH signaling pathway in the EBV-infected cells. Upregulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) was found in almost all EBV positive tumor lines and primary tumors. Importantly, overexpression and activation of NOTCH3 receptor occurred in 6/6 (100%) of NPC tumor lines and 18/26 (70%) of primary tumors. Knockdown of NICD3 expression by siRNA led to significantly reduction of RBP-Jkappa promoter activity and expression of the downstream effectors in the EBV-positive NPC cell line, C666-1. By microarray analysis, a number of targets (e.g. CCND1, C-MYC, and BCL-XL) involved in cell proliferation and survival were identified in the NICD3-silenced NPC cells. Our functional study also demonstrated NICD3 inactivation down-regulated the proliferation of C666-1 cells, both on the viability and DNA synthesis. Increase apoptotic cells were found in the NICD3 siRNA-treated NPC cells. Our data support the hypothesis that NOTCH signaling pathway plays a crucial role in NPC through regulating apoptosis. Since NOTCH signaling is a highly conserved pathway important for human cancers, NICD3 may serve as a potential therapeutic target for treating patient suffering from this EBV-associated cancer. However, we revealed that NPC cells are resistance to gamma-secretase inhibitor (DAPT) treatment. It may be due to the frequent inactivation of NUMB, a negative regulator of NOTCH pathway, in NPC tumors. Importantly, we revealed that NOTCH3 transcription is regulated by CXCR4 in NPC cells. Expression of NOTCH3 was highly reduced in the C666-1 cells treated by either CXCR4 antagonist (AMD3100) or siRNA. In addition to suppression of NICD3 signal, knockdown CXCR4 resulted in growth inhibition and tumor apoptosis. Finally, we demonstrated that the drug resistance of C666-1 towards cisplatin was significantly decreased after CXCR4 antagonist (AMD3100) treatment. Increased tumor apoptosis were detected in the NPC cells treated with both cisplatin and AMD3100 when compared with that treated with cisplatin only. In summary, the CXCR4 antagonist suppresses the NOTCH pathway, inhibits apoptosis signals in NPC cells and thereby enhances its sensitivity to chemotherapeutic treatment. 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 356.

CD26, which is a costimulatory molecule and peptidase, is responsible for the degradation of interferon (IFN)-γ-induced chemokines. To elucidate the immunopathological role of CD26 in allergic asthma, we investigated plasma soluble CD26 (sCD26) concentration and its cell surface expression on lymphocytes, monocytes, CD4+ T helper, CD8+ T suppressor plus cytotoxic T, invariant natural killer T (iNKT), and CD19+ B lymphocytes in allergic asthmatic patients. Plasma sCD26 was significantly elevated in asthmatic patients regardless of inhaled corticosteroid treatment (all P