NPC Cell Research Articles

Ergosta-7,22-diene-2β,3α,9α-triol (EGDT) from Ganoderma lucidum inhibits nasopharyngeal carcinoma cells by blocking EGFR signaling pathway

ObjectiveTo investigate the efficacy and mechanism of EGDT against NPC cell lines. MethodsMTT assay was used to assess cell proliferation inhibition of EGDT. The apoptotic induction and cell cycle arrest were detected by flow cytometry. Western blot was adopted to detect the protein levels. Quantitative Real-time PCR was used to determine the mRNA expressions. The NPC xenografts were established to evaluate the tumor growth inhibition of EGDT. Immunohistochemistry was applied to analyze the EGFR expression in the tumor tissues. ResultsEGDT showed proliferation inhibition on the NPC cell, induced G0/G1 phase arrest and cell apoptosis in vitro. EGDT decreased the protein and mRNA levels of EGFR and its downstream RAF/MEK/ERK and PI3K/AKT pathways in time- and dose-dependent manner. Furthermore, EGDT also showed a sound antitumor activity in NPC xenograft in vivo. ConclusionThe treatment of EGDT displays EGFR and its mediated downstream signaling pathway blockade through decreasing the protein and mRNA levels, suggesting a promising strategy in treating human NPC.

Baicalin hydrate inhibits cancer progression in nasopharyngeal carcinoma by affecting genome instability and splicing.

Baicalin hydrate (BH), a natural compound, has been investigated for many years because of its traditional medicinal properties. However, the anti-tumor activities of BH and its epigenetic role in NPC have not been elucidated. In this study, we identified that BH inhibits NPC cell growth in vivo and in vitro by inducing apoptosis and cell cycle arrest. BH epigenetically regulated genome instability by up-regulating the expression of satellite 2 (Sat2), alpha satellite (α-Sat), and major satellite (Major-Sat). BH also increased the level of IKKα, Suv39H1, and H3K9me3 and decreased LSH expression. Interestingly, BH promoted the splicing of Suv39H1 via the enhancement of m6A RNA methylation, rather than DNA methylation. Taken together, our results demonstrated that BH has an anti-tumor role in NPC and revealed a unique role of BH in genome instability and splicing in response to DNA damage.

Coronarin D induces reactive oxygen species-mediated cell death in human nasopharyngeal cancer cells through inhibition of p38 MAPK and activation of JNK.

Background and PurposeNasopharyngeal carcinoma (NPC) belongs to squamous cell carcinoma that occurs in the epithelial lining of the nasopharynx. Because of the anatomical position close to the cervical lymph node, some patients have a distant metastasis at the time of diagnosis that leads to treatment failure. Although early stages have a high curability and excellent prognosis, advanced NPC urgently requires new drugs developed to reinforce the effectiveness of therapy without noticeable side effects.Experimental approachCoronarin D (CD), a natural product extracted from the rhizomes of Hedychium coronarium, has been reported to possess anticancer potential. The aim of the present study was to determine the anticancer activity of CD and further elucidate the underlying molecular mechanisms.Key ResultsIn this study, we first demonstrated that CD potently suppressed cell viability in various NPC cell lines. Treatment of cells with CD induced G2/M arrest, apoptosis, and autophagy. Further studies showed that CD increased the production of reactive oxygen species and subsequently activated both autophagy and apoptosis. Moreover, we found that CD-induced activation of p38 and JNK constituted major mechanisms involved in the apoptosis and autophagy triggered by CD. In particular, inhibition of autophagy could strengthen the cytotoxicity of CD, implying that autophagy seems to play a valuable survival and protective role in cancer cells.Conclusions & ImplicationsThese findings provide a promise for the use of CD in combination with autophagy inhibitors for treatment of human NPC cell lines.

Downregulation of CCL2 induced by the upregulation of microRNA-206 is associated with the severity of HEV71 encephalitis.

MicroRNAs (miRNAs) have been investigated widely as key regulators of gene expression in different diseases by affecting the miRNA-mediated regulatory function. Human enterovirus 71 (HEV71) can cause a series of human diseases, including encephalitis. Chemokine (C-C motif) ligand 2 (CCL2) is one of the important genes involved in regulating inflammation. However, the mechanisms underlying HEV71 encephalitis mediated by CCL2 remain to be elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction analysis was used to determine the expression level of miR-206 and the mRNA expression of CCL2 in samples. Western blot analysis was used to detect the protein levels of CCL2. A luciferase assay was used to verify the miR-206 target site in CCL2. A CCK-8 assay and flow cytometry were used to determine cell proliferation and apoptosis. The results demonstrated that miR-206 was downregulated in severe HEV71 encephalitis. Using bioinformatics analysis, miR-206 was predicted to target the human CCL2 3′-untranslated region (3′-UTR). A dual-luciferase assay demonstrated that miR-206 downregulated the expression of CCL2 by directly targeting its 3′-UTR, whereas CCL2 3′-UTR mutations completely eliminated its interaction with miR-206. The expression levels of miR-206 and CCL2 were inversely correlated in cerebrospinal fluid. The expression of exogenous miRNA, which mimicked miR-206 miRNA, decreased the protein and mRNA levels of CCL2, whereas the suppression of endogenous miR-206 resulted in an increase of the protein and mRNA levels of CCL2. The present study also found that miR-206 promoted NPC cell proliferation and reduced the apoptosis of NPC cells via CCL2. The mechanism is likely to involve suppression of the expression of miR-206 and upregulation of the expression of CCL2, important in regulating the progress of HEV71 encephalitis. In conclusion, miR-206 may be useful in the prognosis and treatment of HEV71 encephalitis.

Abstract 14: Anticancer activities of terpenoid compounds isolated from the plant centipeda minma against nasopharyngeal carcinoma cells

Abstract Nasopharyngeal carcinoma (NPC) is one of common cancers throughout south-east Asia, southern China and other regions. Radiotherapy and chemotherapy are the standard treatments for NPC with severe adverse effects, multi-drug resistance, and insensitivity to advanced NPC. Complementary and novel treatments for NPC gain considerable interest around the world. The medicinal plant Centipeda minma is traditionally used in China for the treatment of various nasal diseases such as allergy, rhinitis, sinusitis and nasopharyngeal carcinoma. In the current study, we studied the anticancer potential of an ethanol extract (CME) and 3 pure sesquiterpenoids (A1, A2, A3) extracts from Centipeda minma against NPC cells in vitro and some underlying mechanisms of actions. The NPC cell lines CNE-1 and CNE-2 cells were treated with either CME or 3 pure compounds (A1, A2, A3). Proliferation of NPC cells was inhibited in a concentration- and time-dependent manner. For 48 h treatment, the IC50 values were 41.57 to 24.98 μg/mL for CME, and 2.3 to 7.5 μM for A1 to A3. While these extracts showed no appreciable effect on normal human LO2 hepatocytes, significant numbers of NPC cells underwent apoptotic morphological changes following treatments. Cytometric analysis demonstrated that treatments with CME and compounds results in NPC cell cycle arrest at the G2/M phase and eventually apoptosis in a concentration and time-dependent manner. Western blot analysis showed that these extracts could down-regulate Bcl-2 expression, up-regulate Bax expression and caspase-8, 9, 3 activities. In addition, the potential anti-NPC actions might be also associated with the marked inhibition of multiple signal transduction pathways including EGFR, VEGFR, HER2, FGFR, IGF-1R, PI3K/AKT/mTOR, MAP/ERK and Jan/Stat3 pathways. In summary, the Centipeda minma extract and terpenoids are potential apoptosis inducing agents to kill NPC cells and may be promising anti-cancer drug leads against nasopharyngeal carcinoma. The exact mechanism of actions remains further study. Note: This abstract was not presented at the meeting. Citation Format: Bei-bei Liu, Kui-yuan Zhu, Chi-on Chan, Ling-Hua Zhang, Daniel Kam-Wah Mok, Si-bao Chen. Anticancer activities of terpenoid compounds isolated from the plant centipeda minma against nasopharyngeal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 14. doi:10.1158/1538-7445.AM2017-14

RERG suppresses cell proliferation, migration and angiogenesis through ERK/NF-\u03baB signaling pathway in nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a malignancy of the head and neck that is prevalent in Southeast Asia and southern China. Recent studies in epigenetics suggest that DNA methylation plays a pivotal role in the onset and progression of cancer. Combining the methyl-DNA binding domain capture technique and cDNA microarray analysis, we identified a unique hypermethylated gene, RERG (Ras-like estrogen-regulated growth inhibitor), that was down-regulated in NPC tissues. RERG is a tumor suppressor gene that was first reported in breast cancer. However, the functions of RERG are largely unknown in other tumor types.MethodsRERG expression was assessed in human subjects (NPC primary tissues and non-cancer tissues) and cell lines (NPC cell lines and an immortalized epithelial cell line NP460). Further, we investigated the methylation rate of RERG in both human subject and cell lines. 5-Aza-2’-deoxycytidine (Aza) or combined with trichostatin A (TSA) were treated to three NPC cell lines (HK1, C666-1 and HK1_EBV). In addition, the role of RERG in NPC cells and its underlying mechanisms were explored by overexpression of RERG in NPC cell lines.ResultsRERG was significantly down-regulated in NPC cancer nests compared to normal nasopharyngeal epithelium cells. Furthermore, the RERG promoter was frequently methylated in NPC tissues and cell lines. The RERG methylation rate yielded an area under the curve (AUC) of receiver operating characteristic (ROC) curve was 0.897 (95%CI: 0.818–0.976). The down-regulation of RERG was restored in NPC cells treated with Aza and TSA. In addition, ectopic expression of RERG in NPC cell lines resulted in a significant suppression of cell proliferation, clonogenicity, migration and invasion. RERG-overexpressing cells showed significantly slower growth and less angiogenesis in tumor xenografts in nude mice. RERG suppressed the ERK/NF-κB signaling pathway and inhibited tumor growth and angiogenesis with down-regulation of MMPs and IL8 in tumors of nude mouse xenografts.ConclusionsOur results suggest that RERG is frequently silenced by promoter CpG methylation in NPC, and acts as a functional tumor suppressor by suppressing the ERK/NF-κB signaling pathway. These findings support the potential use of RERG as a novel molecular target in NPC therapy.

TIMELESS confers cisplatin resistance in nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway and promoting the epithelial mesenchymal transition

This study investigated the expression, clinicopathological significance and mechanism of action of TIMELESS, a mammalian homolog of a Drosophila circadian rhythm gene, in nasopharyngeal carcinoma. Quantitative real-time PCR, western blotting and immunohistochemistry revealed TIMELESS was upregulated in NPC cell lines (n = 8 vs. NP69 cells), and freshly-frozen (n = 6) and paraffin-embedded human NPC specimens (n = 108 vs. normal samples/non-tumor cells). TIMELESS expression was associated with T category (P = 0.002), N category (P = 0.001), clinical stage (P < 0.001), metastasis (P = 0.047), vital status (P = 0.013) and serum Epstein–Barr DNA (P = 0.005). High TIMELESS expression was associated with poorer overall survival (80.7% vs. 95.9%; P = 0.004) and progression free survival (68.1% vs. 88.0%; P = 0.005). Univariate and multivariate analysis revealed TIMELESS was an independent prognostic factor for overall survival and progression free survival. Stable ectopic overexpression of TIMELESS in NPC cell lines conferred resistance to cisplatin-induced apoptosis in vitro and in vivo, promoted an epithelial-to-mesenchymal transition phenotype, and activated the Wnt/β-catenin pathway and downstream gene transcription; knockdown of TIMELESS had the opposite effects. TIMELESS may play a role in the development of NPC and could represent a valuable prognostic factor and potential therapeutic target.

CDC42-interacting protein 4 promotes metastasis of nasopharyngeal carcinoma by mediating invadopodia formation and activating EGFR signaling

BackgroundNasopharyngeal carcinoma (NPC) is a common malignancy in Southern China and Southeast Asia. In this study, we investigated the functional and molecular mechanisms by which CDC42-interacting protein 4 (CIP4) influences NPC.MethodsThe expression levels of CIP4 were examined by Western blot, qRT-PCR or IHC. MTT assay was used to detect the proliferative rate of NPC cells. The invasive abilities were examined by matrigel and transwell assay. The metastatic abilities of NPC cells were revealed in BALB/c nude mice.ResultsWe report that CIP4 is required for NPC cell motility and invasion. CIP4 promotes the activation of N-WASP that controls invadopodia formation and activates EGFR signaling, which induces downstream MMP2 (matrix metalloproteinase 2) upregulation. In addition, CIP4 could promote NPC metastasis by activating the EGFR pathway. In nude mouse models, distant metastasis was significantly inhibited in CIP4-silenced groups. High CIP4 expression is an independent adverse prognostic factor of overall survival (OS) and distant metastasis-free survival (DMFS).ConclusionWe identify the critical role of CIP4 in metastasis of NPC which suggest that CIP4 may be a potential therapeutic target of NPC patients.

TNFAIP3 inhibits migration and invasion in nasopharyngeal carcinoma by suppressing epithelial mesenchymal transition.

TNF alpha induced protein 3 (TNFAIP3), a member of zinc finger protein family, is a gene whose expression level is promptly induced by the tumor necrosis factor. In this study, the clinical significance of TNFAIP3 was analyzed based on available samples in The Cancer Genome Atlas database. TNFAIP3 downregulation was associated with distant metastasis and worse patient prognosis. TNFAIP3-overexpressing and TNFAIP3-knockdown NPC cell line models were established through plasmid-mediated overexpression and small interfering RNA (siRNA), respectively. Cell migration and invasion capacities were evaluated by wound-healing and transwell assays. Functional studies indicated that TNFAIP3 knockdown promoted migration and invasion, whereas TNFAIP3 overexpression alleviated these functions. Western blot analysis was used to examine protein changes from TNFAIP3 overexpression and knockdown, in which TNFAIP3 promoted the protein expression of E-cadherin and suppressed vimentin expression. Our data suggested that TNFAIP3 inhibited migration and invasion by suppressing epithelial mesenchymal transition in NPC.

Positive regulation of TAZ expression by EBV-LMP1 contributes to cell proliferation and epithelial-mesenchymal transition in nasopharyngeal carcinoma.

The Epstein-Barr virus latent membrane protein 1 (LMP1) is an integral membrane protein. LMP1 has been reported to activate the NF-κB and mitogen-activated protein kinase pathways. However, these effects alone are unable to account for the profound oncogenic properties of LMP1. TAZ is one of the nuclear effectors of Hippo-related pathways and highly expressed in many human tumors. Here, we reported that TAZ was frequently expressed in LMP1-positive nasopharyngeal carcinoma. In NPC cell lines, we showed that LMP1 promoted TAZ expression. Gelsolin is an important inhibitor of TAZ activity. Our studies showed that LMP1 interacted with gelsolin, resulting in inhibition of Lats1/2 phosphorylation and improvement of TAZ stability. Furthermore, we revealed that TAZ is important for LMP1-mediated cell proliferation, cancer stem cell-like properties and epithelial-mesenchymal transition. These findings provide new insights into the carcinogenic roles of LMP1 and contribute to further understanding of its oncogenic mechanism.

HOPX functions as a tumour suppressor in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is generalized term that encompasses a diverse group of cancers that includes tumours of the oral cavity (OSCC), oropharynx (OPSCC) and nasopharynx (NPC). Genetic alterations that are common to all HNSCC types are likely to be important for squamous carcinogenesis. In this study, we have investigated the role of the homeodomain-only homeobox gene, HOPX, in the pathogenesis of HNSCC. We show that HOPX mRNA levels are reduced in OSCC and NPC cell lines and tissues and there is a general reduction of HOPX protein expression in these tumours and OPSCCs. HOPX promoter methylation was observed in a subset of HNSCCs and was associated with a worse overall survival in HPV negative tumours. RNAseq analysis of OSCC cells transfected with HOPX revealed a widespread deregulation of the transcription of genes related to epithelial homeostasis and ectopic over-expression of HOPX in OSCC and NPC cells inhibited cell proliferation, plating efficiency and migration, and enhanced sensitivity to UVA-induced apoptosis. Our results demonstrate that HOPX functions as a tumour suppressor in HNSCC and suggest a central role for HOPX in suppressing epithelial carcinogenesis.

Downregulation of long non-coding RNA MEG3 in nasopharyngeal carcinoma.

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.

LncRNA ANRIL is up-regulated in nasopharyngeal carcinoma and promotes the cancer progression via increasing proliferation, reprograming cell glucose metabolism and inducing side-population stem-like cancer cells.

Long noncoding RNAs play a vital role in diverse biological processes such as embryonic development, cell growth, and tumorigenesis. In this study, we report that LncRNA ANRIL, which encodes a 3834-nt RNA that contains 19 exons at the antisense orientation of the INK4B-ARF-INK4A gene cluster, generally up-regulated in nasopharyngeal carcinoma [1]. In a cohort of 88 NPC patients, ANRIL was highly expressed in advanced-stage cancer. Multivariate analyses revealed that ANRIL expression could serve as an independent predictor of overall survival (P = 0.027) and disease-free survival (P = 0.033). Further investigation showed that knockdown of ANRIL significantly repressed NPC cell proliferation and transformation. We also found that ANRIL could induce the percentage of side population cells (SP cells) in NPC. To meet the urgent needs of energy provision, ANRIL can also reprogram glucose metabolism via increasing glucose uptake for glycolysis, which was regulated by the mTOR signal pathway to affect the expression of essential genes in glycolysis. We concluded that ANRIL could promote NPC progression via increasing cell proliferation, reprograming cell glucose metabolism and inducing side-population stem-like cancer cells. Our results also suggested that ANRIL may serve as a novel diagnostic or prognostic biomarker and a candidate target for new therapies in NPC.

Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom 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 632.

Abstract 2187: Garcinone inhibits nasopharyngeal carcinoma cell growth and arrests cell cycle via modulating the expression of ATR/Stat3/4E-BP1

Abstract Nasopharyngeal carcinoma (NPC) is one of the common head and neck malignancies. Radiotherapy and chemotherapy are the standard treatments for NPC. Garcinone, a natural compound isolated from Garcinia oblongifolia Champ., is a xanthone derivative and has been shown to have potential cytotoxic effects on certain cancer types. However, there have been limited studies performed on its effects on NPC cells and the mechanisms of these effects remain unknown. In this study, we firstly found that garcinone significantly inhibited cell viability of human NPC cell lines CNE1, CNE2, HK1, and HONE1. This inhibition was in a time- and dose-dependent manner. Then, flow cytometry analysis was performed on cell cycle analysis. We observed that garcinone arrested cell cycle at S phase. With 10μM of high-dose garcinone treatment, the cells exhibited necrotic morphology changes including cell swelling, rough endoplasmic reticulum (ER) degranulation, ER dilatation, mitochondria swelling, and vacuolar degeneration. Finally, we found that garcinone stimulated the expression levels of ATR and 4E-BP1, while efficiently inhibited the expression levels of Cyclin B1, Cyclin D1, Cyclin E2, cdc2, CDK7, and Stat3. Collectively, the ability of garcinone to inhibit NPC in vitro suggested that garcinone might be a novel agent for the management of NPC. Supported by NSFC Key Project 81130046; NSFC Project 81171993 and NSFC81272415; Guangxi Key Projects 2013GXNSFEA053004 and 2012GXNSFCB053004; Guangxi Ministry of Education 201202ZD022 and 201201ZD004. Citation Format: Yudui Xia, Xia Liu, Chunllin Zou, Hongwei Guo, Yeguo Yang, Yong Lei, Jian Zhang, Yi Lu. Garcinone inhibits nasopharyngeal carcinoma cell growth and arrests cell cycle via modulating the expression of ATR/Stat3/4E-BP1. [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 2187.

Abstract 4064: The identification of UBR5-ZNF423 recurrent fusion gene in EBV-associated nasopharyngeal carcinoma

Abstract 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.

Abstract 626: Application of patient-derived xenograft model in nasopharyngeal carcinoma research

Abstract Nasopharyngeal carcinoma (NPC) was an Epstein Barr virus (EBV)-related malignancy and tumor microenvironment had a pivotal role in tumor progression. Paucity of good NPC animal models hindered the research in this field. Recently, patient-derived xenograft (PDX) had been shown to be a good preclinical model for drug screening and cancer related research. We had developed two PDX mice lines from engrafting NPC metastatic tumors. Positive EBV-encoded small RNAs staining confirmed these tumors harboring EBV. Further gene expression profile analysis showed higher similarity of PDX to primary parent tumor than NPC cell line xenograft. In vivo drug screening in the PDX system demonstrated gemcitabine had the best antitumor effect among the tested drugs. In this PDX corresponding patient also showed excellent response to gemcitabine treatment. Combination of gemcitabine and valproic acid had synergistic antitumor effect. Further adding ganciclovir in this two combined regimen enhancing cytolytic viral activation had the best antitumor response among the tested regimens. This three combined regimen treated group had lower plasma EBV-DNA load and tumor viral concentration and less viable tumor cells than gemcitabine + valproic acid group. These promising results would open a new era for EBV-targeting therapy in NPC treatment. Citation Format: Cheng-Lung Hsu, Yung-Chia Kuo, Yenlin Huang, Yin-Cheng Huang, Kar-Wai Lui, Kai-Ping Chang, Tung-Liang Lin, Hsien-Chi Fan, An-Chi Lin, Chia-Hsun Hsieh, Li-Yu Lee, Hung-Ming Wang, Hsin-Pai Li, Angel Chao, Yu-Sun Chang. Application of patient-derived xenograft model in nasopharyngeal carcinoma research. [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 626.

Overexpression of PIN1 Enhances Cancer Growth and Aggressiveness with Cyclin D1 Induction in EBV-Associated Nasopharyngeal Carcinoma.

BackgroundNasopharyngeal 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.MethodsThe 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.ResultsWe 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.ConclusionsThis 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.

Twist Expression of Nasopharyngeal Carcinoma Predicts Pre-vertebral Space Invasion and Survival Outcome

BackgroundThe purpose of this study was to explore Twist expression in nasopharyngeal carcinoma and to investigate the prognostic impact of Twist expression on survival rates in patients with nasopharyngeal carcinoma. MethodsWe utilized Western blotting to investigate the expression of Twist, vimentin, and E-cadherin in NPC cell lines. Additionally, a retrospective review of case notes from the Dalin Tzu Chi General Hospital archives was performed. We analyzed the relationship between Twist expression of tissue specimen, TNM staging, and survival rates by using immunohistochemical staining. All nasopharyngeal carcinoma patients were included. We used Pearson's chi-square and Fisher's exact tests to analyze the correlation between Twist expression of tissue specimen and TNM staging and clinical features. Kaplan-Meier survival curves were constructed and survival analysis was completed using Cox proportional hazard models. Receiver operating characteristic (ROC) curve was also examined in order to validate the ability of Twist expression to predict outcomes. ResultsA total of 48 patients with newly diagnosed NPC were enrolled in this study. Nineteen patients (40%) in this series were found to have Twist overexpression. Twist overexpression in NPC patients was associated with pre-vertebral space invasion (P = 0.032). Patients with Twist overexpression were found to have a poor overall survival rate compared with others (P = 0.01, respectively), but not metastasis free-survival rate (P = 0.126). There was a trend toward significance between Twist expression and advanced T classification (P = 0.094 respectively). Multivariate analysis showed that Twist overexpression was associated with poor survival rate (hazard ratio 5, 95% confidence interval 1.07-27; P = 0.041). The receiver operating characteristic (ROC) curves for predicting outcomes revealed that Twist expression (area under curve = 0.735) was superior to T classification (area under curve = 0.718) and clinical stage (area under curve = 0.598). ConclusionsTwist may be a useful molecular marker for nasopharyngeal carcinoma and indicator for overall survival. Patients with Twist expression should be treated more aggressively.

S100A4 hypomethylation affects epithelial-mesenchymal transition partially induced by LMP2A in nasopharyngeal carcinoma.

To identify cellular target genes involved in NPC cell invasion and metastasis, gene expression profiles of CNE-1 cells with or without ectopic LMP2A expression were compared by using the metastatic gene array. S100 calcium binding protein A4 (S100A4) was the highest increased one among these genes both in mRNA and protein levels of NPC cells. Moreover, S100A4 was upregulated in LMP2A-positive NPC tissues. We found that CNE-1-S100A4 showed significantly increased invasion ability as compared to the controls both in vitro and in vivo, which indicated that S100A4 induced EMT occurrence and promoted metastasis. Notably, the DNA hypomethylation of S100A4 was found in LMP2A-positive NPC tissues. Besides, inhibition of DNA methyltransferases via 5-Aza-dC stimulated the expression of S100A4 in the cells without ectopic LMP2A expression. The methylation changes were confirmed by methylation specific PCR (MSP), suggesting that LMP2A ectopic expression led to the demethylation of S100A4 promoter. These results demonstrated that LMP2A-induced hypomethylation participated in regulating S100A4 expression in NPC. Our findings provide an evidence for the emerging notion that hypomethylation and activation of correlated genes are crucial for metastasis progression in cancer. © 2015 Wiley Periodicals, Inc.