NPC Cell Research Articles

Abstract 2483: Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression

Abstract Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy. Citation Format: Bok-Soon Lee, Sam Kang, Kyung-Ah Kim, Yun-Jeong Song, Kwang Ho Cheong, Hyun-Young Cha, Haeng-Jun Kim, Hye Sook Hwang, Yeon Soo Kim, Chul-Ho Kim. Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression. [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 2483. doi:10.1158/1538-7445.AM2015-2483

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

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

Abstract 1753: XIAP maintains the characteristics of cancer stem cells and is a therapeutic target in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common malignancy in South China. Cancer stem cells (CSCs) are thought as the key players in the resistance to chemo- or radio-therapy and are response for tumor recurrence and metastasis, while their ability to escape from the apoptosis pathway may render them the resistant property to the therapies. We show that XIAP is overexpressed in the NPC cell line S18, which have the higher SP populations and CD44+ populations and the stronger abilities of sphere-formation and metastasis. Importantly, XIAP knockdown decreases SP and CD44+ populations, prevents sphere-formation and reduces the ability of invasion and migration. What more, loss of XIAP enhances the sensitivity of cells to conventional chemotherapy drugs CDDP and 5-FU. The primary xenograft tumor formation in vivo found there is a dramatic reduction in tumor formation in the XIAP KD group. They all indicate that XIAP plays an important role in nasopharyngeal cancer stem cell. Further studies show that XIAP limits MAPK signaling and thereby restricts differentiation and maintains the characteristics of cancer stem cells. We designed and synthesized a novel Smac mimetic compound APG-1387 that promotes the rapid degradation of cIAP1/2 and XIAP, and it exerts an antitumor effect on nasopharyngeal carcinoma cancer stem cells. Further studies show that APG-1387 enhances the chemosensitivity and promotes apoptosis in combination with CDDP and 5-FU of NPC in vitro and vivo. Citation Format: jiao ji, Rong Deng, Wen-Dan Chen, Gong-Kan Feng, Xiao-Feng Zhu, Da-Jun Yang. XIAP maintains the characteristics of cancer stem cells and is a therapeutic target in nasopharyngeal carcinoma. [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 1753. doi:10.1158/1538-7445.AM2015-1753

Protocadherin20 Acts as a Tumor Suppressor Gene: Epigenetic Inactivation in Nasopharyngeal Carcinoma.

Genetic alterations of 13q21 (PCDH 8,9,17, and 20) are frequently observed in multiple tumors, suggesting the presence of critical tumor suppressor genes (TSGs). Protocadherin20 (PCDH20), located at 13q21.2, belongs to the δ1-protocadherins, which constitutes one of the largest subgroup within the adherin superfamily. Frequent downregulation/silencing of PCDH20 was found in NPC cell lines using semiquantitative PCR. PCDH20 mRNA was broadly expressed in normal nasopharyngeal tissues and cell lines. Promoter methylation of PCDH20 was observed in 80% (4/5) of NPC cell lines and 78.4% (40 of 51) of primary tumors by methylation-specific PCR, but rarely in normal nasopharygeal tissues and nasopharyngeal epithelial cell line (NP69). The silencing of PCDH20 can be reversed by pharmacological demethylation, indicating an epigenetic mechanism. Restoration of PCDH20 expression in NPC cells strongly suppressed cell numbers and colony formation. Overexpression of PCDH20 antagonized Wnt/β-catenin signaling pathway and promoted β-catenin to translocate from nucleus to cytoplasm and membrane. PCDH20 significantly inhibited the migration and invasion ability of NPC cells. PCDH20 also inhibited epithelial-mesenchymal transition (EMT) through enhanced expression of E-cadherin. Our study identified PCDH20 as a functional tumor suppressor and an important antagonist of Wnt/β-catenin signaling and EMT, with frequent epigenetic inactivation in NPC.

Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells.

Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced γH2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 μM) of the topoisomerase II inhibitor etoposide. Regulation of γH2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, including ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells. NPC is characterized by clonal EBV infection and accounts for >78,000 annual cancer cases with increased incidence in regions where EBV is endemic, such as southeast Asia. The latent proteins LMP1 and LMP2A coexpressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A coexpression suppressed activation of the DNA damage response (DDR) protein γH2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A-coexpressing cells and indicating distinct LMP1-dependent effects in the restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the cooperative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

LOC401317, a p53-regulated long non-coding RNA, inhibits cell proliferation and induces apoptosis in the nasopharyngeal carcinoma cell line HNE2.

Recent studies have revealed that long non-coding RNAs participate in all steps of cancer initiation and progression by regulating protein-coding genes at the epigenetic, transcriptional, and post-transcriptional levels. Long non-coding RNAs are in turn regulated by other genes, forming a complex regulatory network. The regulation networks between the p53 tumor suppressor and these RNAs in nasopharyngeal carcinoma remains unclear. The aims of this study were to investigate the regulatory roles of the TP53 gene in regulating long non-coding RNA expression profiles and to study the function of a TP53-regulated long non-coding RNA (LOC401317) in the nasopharyngeal carcinoma cell line HNE2. Long non-coding RNA expression profiling indicated that 133 long non-coding RNAs were upregulated in the human NPC cell line HNE2 cells following TP53 overexpression, while 1057 were downregulated. Among these aberrantly expressed long non-coding RNAs, LOC401317 was the most significantly upregulated one. Further studies indicated that LOC401317 is directly regulated by p53 and that ectopic expression of LOC401317 inhibits HNE2 cell proliferation in vitro and in vivo by inducing cell cycle arrest and apoptosis. LOC401317 inhibited cell cycle progression by increasing p21 expression and decreasing cyclin D1 and cyclin E1 expression and promoted apoptosis through the induction of poly(ADP-ribose) polymerase and caspase-3 cleavage. Collectively, these results suggest that LOC401317 is directly regulated by p53 and exerts antitumor effects in HNE2 nasopharyngeal carcinoma cells.

Abstract 5003: Functional significance of TIGAR expression in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus associated cancer of the nasopharynx. Annual incidence in China is high, reaching up to 25 per 100,000. NPC is a highly invasive and metastatic disease. At diagnosis, over 60% of patients have advanced disease where treatment failure due to distant recurrence or metastasis is common. More effective forms of therapy to treat this deadly disease are therefore urgently needed. The TP53 induced glycolysis and apoptosis regulator (TIGAR) is a p53 target gene which blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway. TIGAR promotes production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), leading to enhanced scavenging of intracellular reactive oxygen species (ROS) and inhibition of oxidative-stress induced apoptosis in normal cells. Previously, we have found that a novel nucleoside analog inhibited cell growth and induced apoptosis in NPC cell lines via down-regulation of TIGAR expression. Moreover, the growth inhibitory effect of c-Met tyrosine kinase inhibitors was ameliorated by over-expression of TIGAR in NPC cell lines. These results are indicative of a pivotal role for TIGAR expression in maintaining NPC cell survival. In this study, we investigated the expression pattern of TIGAR in NPC tumor tissues and the consequences of TIGAR overexpression and knockdown on NPC cell growth and invasion. It was found that TIGAR is overexpressed in 27 out of 36 (75%) NPC cases in the tumor cells compared to their respective normal epithelial cells. Overexpression of TIGAR in NPC cell lines resulted in increased proliferation, invasion, NADPH production and up-regulation of mesenchymal markers including fibronectin and vimentin. These changes were reversed when TIGAR expression was knocked down by transfection with siRNA, demonstrating specificity of the observed effects. Together, our results indicate that TIGAR expression promotes proliferation, invasiveness and expression of mesenchymal markers in NPC cells. These findings may form the basis for development of a new therapeutic target for treatment of NPC in the future. Citation Format: Elaine YL Wong, SC Cesar Wong, Charles ML Chan, Emily KY Lam, Louisa Ho, Cecilia PY Lau, Thomas CC Au, Amanda KC Chan, CM Tsang, George SW Tsao, Vivian WY Lui, Anthony TC Chan. Functional significance of TIGAR expression in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5003. doi:10.1158/1538-7445.AM2014-5003

Abstract 5356: The role of VNN3 gene in nasopharyngeal carcinoma tumorigenesis

Abstract Nasopharyngeal carcinoma (NPC) is one of the leading cause of cancer-related deaths in southeastern China and South East Asia. However, the detailed pathogenesis mechanisms of NPC remain to be elucidated. Despite novel therapies and advances in early detection, it is often diagnosed at an advanced stage and has a poor prognosis. Recent evidences showed that SRY (sex-determining region Y)-box 5 (SOX-5) plays an important role in Nasopharyngeal tumor development, but little is known about the role of its associated protein such as VNN3 gene (a pantetheinase) in controlling tumor development. SOX-5 overexpression enhances tumor formation in NPC, and has a poor prognosis. In this study, we compared the differential gene expression in NPC cell lines and normal nasal mucosal epithelial cells by cDNA microarray analysis, and observed that the RNA and protein expression levels of SOX-5 and VNN3 were dramatically increased in NPC cell lines than normal nasal mucosal epithelial cells by qRT-PCR and Western blot analysis. In order to investigate that whether VNN3 gene could alter NPC progression, we observed at first the gene expression in different NPC cell lines using qRT-PCR analysis and found that the expression of VNN3 in NPC cell line, especially in NPC-TW01 cell line was highly upregulated. Besides, we also found that the expression level of VNN3 protein was also increased in multiple NPC cell lines and NPC biopsy specimens by immunohistochemical staining and Western bloting. In this study, we also isolated the cancer stem cell (CSC) from NPC-TW01 cell line by conditioned culture medium, highly expression level of VNN3 gene was found in the NPC-TW01 stem cells (CSC). To further analysis of the functional role of VNN3 in this NPC and CSC lines, the expression of VNN3 was decreased to about 70-80% of the control in NPC and CSC after knockdown of VNN3 by VNN3 shRNA-lentiviral infection. This in turn resulted in a significant reduction of cell proliferation, migration and invasion abilities. We also found that a decreased expression level of VNN3 (Pantetheinase), which is an amidohydrolase that hydrolyzes pantetheine into pantothenic acid (vitamin B5) as well as cysteamine, and caused an increase of intracellular reactive oxygen species (ROS), and cell death through apoptosis; the weight of NPC xenograft tumors had diminished about 51% after 6 weeks, compared with shLuc control in SCID mice bearing NPC xenografts derived from these lentiviral infected NPC and CSC cell lines. Impairment of tumor cell proliferation, accompanied with apoptosis was found in the xenografts histopathologically. These novel findings of the functional role of VNN3 may have a potential implication in molecular targeted therapy for NPC. Citation Format: Shang-Yu Huang, Yi-Ying Chen, Dah-yeou Huang, Cheng-Der Wu, Chin-Tarng Lin. The role of VNN3 gene in nasopharyngeal carcinoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2014-5356

Abstract 3425: Whole-transcriptome analyses of EBV-associated nasopharyngeal carcinoma using next-generation transcriptome sequencing

Abstract Nasopharyngeal carcinoma (NPC) is unique among epithelial cancers arising from the head and neck regions. This specific cancer type is consistently associated with EBV infection. Using RNA-seq, we have conducted a comprehensive assessment of EBV and cellular gene expression in six NPC cell line and patient-derived xenografts (PDXs). In these tumor lines, expression of EBV latent genes including EBNA1, LMP1, LMP2A and BamH1 A transcripts was quantitated. Among the viral genes, the BamH1 A transcripts were detected as the major transcripts with various splicing variants. Expression of these transcripts in NPC was further validated by qRT-PCR analysis. Active lytic gene expression and lytic viral replication was not observed in these tumor lines. Analysis of the RNA-seq data has identified multiple fusion transcripts. A total of 18 candidate fusions were validated in these tumor lines. A novel fusion transcript, UBR5-ZNF423 was detected in C666-1 cells and 8.3% of primary tumors. The transcript produces a chimeric fusion protein which might alter the function of EBF transcription factor. A significant growth inhibitory effect was observed in UBR5-ZNF423 knockdown C666-1 cells. Constitutive expression of UBR5-ZNF423 fusion in NIH3T3 cells induced in vivo tumor formation in nude mice model. The findings strongly suggest that the fusion gene play a causative role in NPC tumorigenesis. Using the expression profile of immortalized nasopharyngeal epithelial cells as control, our study also revealed the differential expression of cellular genes and specific alternative spliced transcripts in NPC. Significant upregulation of genes involved in cytokine-cytokine receptor interaction (e.g. CCL5, CCL20, CXCL10, CXCR4, IL2RG) was consistently detected in the tumor lines. Most of them were targets of the NF-kappaB signaling pathways, which are constitutively activated in EBV-associated NPC. In addition, the analyses also discovered several potential druggable targets (e.g. CD74) and oncogenic non-coding RNAs (e.g. AFAP1-AS1) in NPC, which may contribute to the development of new therapeutic strategies for this cancer. The whole-transcriptome analyses greatly enhance our understanding of the molecular basis of EBV-infected NPC. Citation Format: Cathy Ka-Yan Mak, Grace Tin-Yan Chung, Kevin Yuk-Lap Yip, Ken Kai-Yuen Tso, Sau-Dan Lee, Siu-Tim Cheung, Sai-Wah Tsao, Pierre Busson, Ka-Fai To, Kwok-Wai Lo. Whole-transcriptome analyses of EBV-associated nasopharyngeal carcinoma using next-generation transcriptome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2014-3425

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

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

Effect of SOX1 gene overexpression on chemotherapeutic sensitivity of nasopharyngeal carcinoma cells

Objective To explore the effect of overexpressed SOX1 gene upon chemotherapeutic sensitivity of nasopharyngeal carcinoma cells.Methods Via Lipofectamin2000,SOX1 gene was transfected into NPC cell lines CNE2 and HONE 1.Cellular SOX1 expression was detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot analysis.MTT was employed to investigate the sensitivity of DDP treatment between NPC cells with or without SOX1 overexpression.Results RT-PCR and Western blot results demonstrated that SOX1 mRNA and protein level of CNE2-SOX1 and HONE1-SOX1 cells were significantly higher than the two NPC cell lines in control group.MTT result showed that NPC cell lines which overexpressed SOX1 were more sensitive to DDP treatment than that in control group (P ＜ 0.05).Conclusion Transfection of extrinsic SOX1 gene contributes to its overexpression in CNE2 and HONE1,which can increase the chemotherapeutic sensitivity of the two NPC cell lines. Key words: Nasopharyngeal Carcinoma; SOX1; Chemotherapeutic sensitivity

Effect of WNT5A on epithelial-mesenchymal transition and its correlation with tumor invasion and metastasis in nasopharyngeal carcinoma

ObjectiveTo investigate the correlation between nasopharyngeal carcinoma cell WNT5A and epithelial-mesenchymal transition (emt)/metastasis, and investigate its possible mechanisms. MethodsRT-PCR and gene transfection were used to detect the expression of nasopharyngeal carcinoma cell strains WNT5A and EMT related factor 5–8F. Transient transfection of NPC cell line 5–8F was determined by liposome of plasmid with WNT5A gene. The differential expressions of WNT5A and EMT-related factors in cells before and after transfection were detected by RT-PCR. Cell scratch assay and Transwell assay were used to detect the motility abilities of cells before and after 5–8F transfection. ResultsThe expressions of WNT5A and EMT related factors matrix metalloproteinase-2 of the WNT5A transferred group in the nasopharyngeal carcinoma cell line 5–8F were higher than the blank control group and the empty vector transferred group, and the transfer ability of the WNT5A transferred group was higher than that in the blank control group and the empty vector transferred group, while the expressions of EMT related factors E-cadherin were lower than that in the blank control group and the empty vector transferred group, and the transfer ability of the WNT5A transferred group was higher than that in the blank control group and the empty vector transferred group. ConclusionsIn nasopharyngeal carcinoma cells, WNT5A can regulate the epithelial-mesenchymal transition and affect the ability of tumor invasion and metastasis of nasopharyngeal carcinoma.

P0127 Curcuminoids as putative ebv lytic induction agents for adjuvant treatment in EBV-positive carcinoma

Background Epstein-Barr virus (EBV) is causally involved in various lymphoid and non-lymphoid cancers, including nasopharyngeal (NPC) and stomach carcinomas (EBVaGC). NPC is endemic in south east Asia, including Indonesia. EBVaGC accounts for about 10% of GC worldwide. We recently initiated efforts to target EBV inside tumour cells by cytolytic virus activation therapy (CLVA), currently being validated in a phase 1/2 trial. We aim to develop oral drugs to improve CLVA treatment availability for low-income countries. Curcumin (diferuloylmethane) from Curcuma longa is used as spice in Asian countries without any toxic effects and has anti-cancer activity. Several curcumin analogues (curcuminoids), developed to increase efficacy and bioavailability, are currently being tested in clinical studies. Curcuminoids affect multiple pathways, including inhibition of COX2 and NF-kB and have potential as oral CLVA regimens for EBV-positive carcinoma. Methods We investigated EBV-reactivating effects of curcuminoids on EBV-positive NPC (C666.1) and EBVaGC cell lines (SNU179 and AGS-BX1). EBV-negative GC (AGS) line was used as a negative control. Standard CLVA, SAHA, and bortezomib were used as positive controls. EBV lytic (Zebra, EA-D) activity was measured by semi-quantitative immunoblot, FACS and indirect immunofluorescence. Effects on cell viability were measured by MTT assay. Findings Curcumin induced EBV lytic activity in AGS-BX1 cells but had marginal effect on natural EBV lines (C666.1,SNU179). However, several curcumin analogues induced EBV reactivation in all three EBV-positive cell lines in a dose- and time-dependent manner. MTT assay showed curcuminoids to be more toxic to EBVaGC cells than to C666.1 cells. Noteworthy curcuminoids showed synergistic effects with current lytic induction agents. Interpretation Curcumin analogues serve as non-toxic drugs to reactivate EBV from latently infected NPC and GC cells, but seem to reactivate EBV differently in NPC versus GC. Curcuminoids at pharmacologically relevant concentrations may promote cytolysis of EBV-positive carcinoma when combined with CLVA drugs. The molecular mechanisms of EBV lytic cycle induction by curcuminoids will be studied to gain further insights in EBV reactivation from latency in different carcinoma models.

RETRACTED ARTICLE: Identification of novel signaling components in N,N’-Dinitrosopiperazine-mediated metastasis of nasopharyngeal Carcinoma by quantitative phosphoproteomics

BackgroundNasopharyngeal carcinoma (NPC) is a highly invasive and metastatic cancer. N,N’-dinitrosopiperazine (DNP), a carcinogen with specificity for nasopharyngeal epithelium, facilitates NPC metastasis. However, the underlying mechanism is not known.MethodsQuantitative phosphoproteomics, using stable isotope labeling of amino acids in cell cultures, was employed to identify phosphoproteins associated with NPC metastasis mediated by DNP. NPC cell line 6-10B, which is relatively less metastatic, was used to investigate DNP-mediated metastasis. Boyden chamber invasion assay was used to measure DNP-induced motility and invasion, and nude mice were used to verify DNP-mediated metastasis in vivo. Several different phosphoproteins detected by proteomics analysis were verified by immunoblotting. DNP-mediated metastasis facilitated by lysine-rich CEACAM1 co-isolated protein (LYRIC) phosphorylation at serine 568 was confirmed using mutations targeting the phosphorylation site of LYRIC. DNP-mediated metastasis through LYRIC phosphorylation was confirmed in the NPC cell line CNE1. DNP-mediated LYRIC phosphorylation at serine 568 was also verified in metastatic tumors of BABL/c nude mice.ResultsBoyden chamber invasion assay indicated that DNP mediated cell motility and invasion of NPC cell 6-10B in vitro, and experiments with nude mice indicated that DNP increased 6-10B metastasis in vivo. In the phosphoproteomics analysis, we detected 216 phosphorylation sites on 130 proteins; among these, 48 phosphorylation sites on 30 unique phosphopeptides were modulated by DNP by at least 1.5-fold. DNP mediated the expression of phosphorylated GTPase, ferritin, LYRIC, and RNA polymerase, and it decreased the expression of phosphorylated torsin-1A protein 1. Furthermore, DNP induced LYRIC phosphorylation at serine 568 to facilitate cell motility and invasion, whereas DNP-mediated motility and invasion was decreased when serine 568 in LYRIC was mutated. In another NPC cell line, CNE1, DNP also mediated cell motility and invasion followed by enhanced phosphorylation of LYRIC at serine 568. Finally, phosphorylated-LYRIC expression at serine 568 was significantly increased in metastatic tumors induced by DNP.ConclusionDNP regulates multiple signaling pathways through protein phosphorylation, including the phosphorylation of LYRIC at serine 568, and mediates NPC metastasis. These findings provide insights on the complexity and dynamics of DNP-facilitated metastasis, and may help to gain a better understanding of the mechanisms by clarifying NPC-induced metastasis.

Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor

BackgroundNasopharyngeal carcinoma (NPC) has a high incidence rate in Southern China. Although there are conventional therapies, the side effects and toxicities are not always tolerable for patients. Recently, the tumoricidal effect of ginsenosides on different cancer cells has been studied. This study aims to investigate the anti-cancer effect of ginsenosides on NPC cells and their underlying mechanism.MethodsThe cytotoxicity of ginsenosides on NPC cell line HK-1 was measured by MTT assay. Apoptosis was detected by propidium iodide staining followed by flow cytometry. A xenograft tumor model was established by injecting nude mice with HK-1 cells. The activation of caspases and apoptosis-inducing factor (AIF) were evaluated by Western blot analysis. Nuclear translocation of AIF was also studied by immunofluorescence staining. Mitochondrial membrane potential was measured by JC-1 dye using flow cytometry.ResultsFour ginsenosides, 20 (S)-Rh2, compound K (CK), panaxadiol (PD) and protopanaxadiol (PPD), induced apoptotic cell death in HK-1 cells in a concentration-dependent manner. CK inhibited HK-1 xenograft tumor growth most extensively and depleted mitochondrial membrane potential depolarization and induced translocation of AIF from cytoplasm to nucleus in HK-1 cells. In addition, depletion of AIF by siRNA abolished CK-induced HK-1 cell death.ConclusionGinsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo.

Indole-3-Carbinol Inhibits Nasopharyngeal Carcinoma Growth through Cell Cycle Arrest In Vivo and In Vitro

Nasopharyngeal carcinoma is a common malignant tumor in the head and neck. Because of frequent recurrence and distant metastasis which are the main causes of death, better treatment is needed. Indole-3-carbinol (I3C), a natural phytochemical found in the vegetables of the cruciferous family, shows anticancer effect through various signal pathways. I3C induces G1 arrest in NPC cell line with downregulation of cell cycle-related proteins, such as CDK4, CDK6, cyclin D1 and pRb. In vivo, nude mice receiving I3C protectively or therapeutically exhibited smaller tumors than control group after they were inoculated with nasopharyngeal carcinoma cells. The expression of CDK4, CDK6, cyclin D1 and pRb in preventive treatment group and drug treatment group both decreased compared with the control group. We conclude that I3C can inhibit the growth of NPC in vitro and in vivo by suppressing the expression of CDK and cyclin families. The drug was safe and had no toxic effects on normal tissues and organs.

Quantitative phosphoproteomic analysis identifies activation of the RET and IGF-1R/IR signaling pathways in neuroblastoma.

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

Abstract B238: PIN1 (Peptidyl-prolyl cis/trans-isomerase, NIMA-interacting 1) is a potential therapeutic target for EBV-associated nasopharyngeal carcinoma.

Abstract Nasopharyngeal carcinoma (NPC) is a common EBV-associated human malignancy in Southern China. NPC is highly radiosensitive and radiotherapy is the mainstay treatment for the patients. However, poor clinical outcome remains the major concern in NPC patients with advanced stage disease. Development of new clinical interventions to treat the disease are importance for control this cancer. PIN1 is a highly conserved enzyme that isomerizes specific phosphorylated Ser/Thr-Pro bonds in multiple proteins. It induces conformational changes and modulates functions of many proteins that promote oncogenesis. We have recently confirmed the high PIN1 expression in almost all NPC primary tumours. The aim of this study was to investigate the potential of PIN1 as a therapeutic target for the treatment of NPC. The oncogenic function of PIN1 was investigated in the EBV-positive NPC cell line C666-1 and an immortalized NP epithelial cells NP69. The antitumor effect of PIN1 inhibition was then studied in both in vitro and in vivo NPC models by using the PIN1 inhibitor juglone. In the stable PIN1-expressing NP cells, we found the upregulation of cyclin D1 and activation of MAPL/JNK pathway. Furthermore, we also demonstrated the interaction between PIN1 and EBNA1 in the NPC cells. PIN1 was also shown to interfere the transcription activity of EBNA1 through binding to the phosphorylation sites of EBNA1. Notably, knockdown of PIN1 by siRNA significantly inhibited cell proliferation, DNA synthesis and colony formation of NPC cells. Treatment with juglone demonstrated significant induction of apoptosis and suppression of cyclin D1 expression in C666-1 cells. The IC50 of C666-1 was approximately 6 μM while that of EBV-negative NPC cells HK-1was 10 μM. A dose-dependent inhibition of tumor growth was observed in mice treated with juglone. A significantly growth suppression and cyclin D1 downregulation was found in the tumours of the mice treated with 1-1.5 mg/kg juglone. In conclusion, our findings imply that targeting Pin1 may serve as a potential therapeutic approach for treating patients suffering from this EBV-associated cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B238. Citation Format: Meng Xu, Chit Chow, Chartia Ching Mei Cheung, Grace Tin-Yun Chung, Kwok Wai Lo. PIN1 (Peptidyl-prolyl cis/trans-isomerase, NIMA-interacting 1) is a potential therapeutic target for EBV-associated nasopharyngeal carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B238.

A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation

BackgroundNasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1.ResultsOur results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts.ConclusionAT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC.

Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1

BackgroundNasopharyngeal carcinoma is a distinct type of head and neck cancer which is consistently associated with Epstein-Barr virus (EBV). The C666-1 cell line is the only in vitro native EBV-infected NPC cell model commonly used for study of the viral-host interaction. Nevertheless, the complete EBV genome sequence in this in vitro EBV-infected NPC model has not been characterized.ObjectiveTo determine the complete EBV genome sequence in C666-1 cells.MethodsThe C666-1 genome was sequenced by 100-bases pair-end massive parallel sequencing. Bioinformatics analysis was performed to extract the EBV sequences and construct an EBV consensus sequence map. PCR amplification and Sanger DNA sequencing were used for sequence validation and gap filling. A phylogenetic analysis of EBV strain in C666-1 cells and other reported EBV strains was performed.ResultsA 171,317 bp complete EBV genome of C666-1 was successfully constructed (GenBank accession number: KC617875). Phylogenetic analysis of EBV genome in C666-1 revealed that the C666-1 EBV strain is closely related to the reported strains in NPC primary tumors.ConclusionC666-1 contains a representative NPC-associated EBV genome and might serve as an important model for studying the roles or function of viral proteins in NPC tumorigenesis.