Increased Expression of Flotillin-2 Protein as a Novel Biomarker for Lymph Node Metastasis in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a head and neck malignant tumor rare throughout most of the world but common in Southeast Asia, especially in Southern China. Flotillin-2 (Flot-2) is not only an important component of cellular membrane, but also involves in various cellular processes such as membrane trafficking, T cell and B cell activation, regulation of several signaling pathways associated with cell growth and malignant transformation, keeping structure and junction of epidermal cells and formation of filopodia. Although such molecular effects of Flot-2 have been reported, whether the expression of Flot-2 protein is associated with clinicopathologic implication for NPC has not been reported. The purpose of this research is to investigate the expression of Flot-2 protein in NPC and control nasopharyngeal epithelial tissues by immunohistochemistry and elucidate the association between the expression of Flot-2 protein and clinicopathological characteristics of NPC. The results showed that the positive percentage of Flot-2 expression in the NPC, nasopharyngeal epithelia with atypical hyperplasia and in the control nasopharyngeal mucosa epithelia was 88.8% (119/134), 76.9% (10/13) and 5.7% (5/88), respectively. There was significantly higher expression of Flot-2 protein in NPC and nasopharyngeal epithelia with atypical hyperplasia compared to the control nasopharyngeal mucosa epithelia (P<0.001, respectively). The positive percentage of Flot-2 protein expression in NPC patients with lymph node metastasis was significantly higher than those without lymph node metastasis. Increasing of Flot-2 expression was obviously correlated with clinical stages of NPC patients. The expression of Flot-2 was proved to be the independent predicted factor for lymph node metastasis by multivariate analysis. The sensitivity of Flot-2 for predicting lymph node metastasis of NPC patients was 93%. Taken together, our results suggest that the increased expression of Flot-2 protein is a novel higher sensitivity biomarker that can predict lymph node metastases in NPC.     

鼻咽癌中VEGF-C的表达及其与颈淋巴结转移的关系

目的探讨血管内皮生长因子C(vascular endothelial growth factor C,VEGF-C)在鼻咽癌组织及其淋巴结转移灶中的表达及意义。方法采用免疫组织化学SP法检测45例鼻咽癌组织及其28例相应的淋巴结转移灶中VEGF-C的表达。结果VEGF-C在鼻咽癌组织及其淋巴结转移灶中的阳性率分别为42.22%和78.57%,两组间比较差异有统计学意义(P<0.05)。VEGF-C在有淋巴结转移的鼻咽癌组织中的表达(57.14%)明显高于无淋巴结转移组(11.76%),差异有统计学意义(P<0.05)。结论VEGF-C的表达可能与鼻咽癌的淋巴结转移密切相关。     

MicroRNAs: Potential diagnostic markers and therapeutic targets for EBV-associated nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a highly malignant cancer with local invasion and early distant metastasis. NPC is highly prevalent in the Southern China and South-eastern Asia. The genetic susceptibility, endemic environment factors, and Epstein-Barr virus (EBV) infection are believed to be the major etiologic factors of NPC. Once metastasis occurs, the prognosis is very poor. It is urgently needed to develop biomarkers for early clinical diagnosis/prognosis, and novel effective therapies for nasopharyngeal carcinoma. In this paper, we systematically reviewed the current progress of miRNA studies in NPC. It has been shown that both host encoded miRNAs and EBV encoded miRNAs play key roles in almost all the steps of epithelia cell carcinogenesis, including epithelial-mesenchymal to stem-like transition, cell growth, migration, invasion, and tumorigenesis. More importantly, some miRNAs could be secreted out and play a role in the microenvironments. The level of sera miRNAs is correlated with the copy numbers of host miRNAs in tumor biopsies. Promising results of gene therapy have been also achieved by lentiviral delivered miRNAs. Taken together, cell free miRNAs would be potential biomarkers of early clinical diagnosis/prognosis; while some miRNAs could be further developed into therapeutic agents in the future.     

Identificating cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.     

E-cadherin 在鼻咽癌组织中的表达及临床意义*

摘要目的：检测鼻咽癌组织中上皮细胞钙黏蛋白（E-cadherin）的表达情况,探讨E-cad 与肿瘤侵袭、转移的关系。方法：收集临床 确诊的存档鼻咽低分化鳞癌石蜡标本40例,鼻炎标本20 例。将每个标本的4 长切片分别进行HE染色、免疫组化PV 二步法及 阴性对照。HE 确认病理类型,结合临床资病例料进行TNM分期,根据PV 二步法染色结果检测E-cadherin 的表达,所得结果用 SPSS17.0 进行统计学检验。结果：E-cadherin 在鼻咽癌组织对比中呈不同程度的降低（P=0.002）,与性别、年龄无明显相关,与T 分 期（P=0.023）、淋巴结转移（P=0.001）、TNM 分期（P=0.000）显著相关。结论：1：E-cadherin 在鼻咽癌组和对照组的表达有显著的差 别,可能参与了鼻咽癌的发生发展。2：E-cadherin 的表达与肿瘤的淋巴结转移和病理分期有相关性,但与年龄和性别无相关性。 E-cadherin 的表达缺失是肿瘤远处转移的重要指标。3：E-cadherin 表达水平可能作为肿瘤侵袭,预测鼻咽癌颈部淋巴结隐匿性转 移的潜在肿瘤标志物。     

Clinical predictors of right upper paraesophageal lymph node metastasis from papillary thyroid carcinoma

ABSTRACT: BACKGROUND: Central and lateral lymph node metastases are quite common in patients with papillary thyroid carcinoma, and the predictors for those metastases have been well studied. Right upper paraesophageal lymph node metastasis has rarely been studied. The aim of this study was to identify the clinicopathological characteristics that may be risk factors for right upper paraesophageal lymph node metastasis in patients with papillary thyroid carcinoma. METHODS: This was a prospective observational study of 243 patients with papillary thyroid carcinoma (PTC) who underwent total thyroidectomy and comprehensive central lymph node dissection with or without lateral lymph node dissection between April 2008 and January 2010. The clinicopathologic findings from these patients were investigated and the patterns of lymph node metastasis were analyzed in the patients who had right upper paraesophageal lymph node disease. RESULTS: Of the 243 patients undergoing lymph node dissection, 14 had right upper paraesophageal lymph node metastases. Two of these patients had right upper paraesophageal lymph node metastasis only, without central compartment metastasis. Univariate analysis of clinicopathologic findings showed that right upper paraesophageal lymph node metastasis had significant association with larger primary tumors, multifocal tumors, extrathyroid extension, and lymphatic invasion (p <0.05 for each factor). CONCLUSIONS: Although there were no independent predictors of right upper paraesophageal lymph node metastasis, it can be the only site of metastasis without other compartmental metastasis. Therefore, during surgery for patients with central or lateral lymph node metastases from PTC, it may be helpful to examine the right upper paraesophageal lymph nodes.     

E-cadherin在鼻咽癌组织中的表达及临床意义

目的：检测鼻咽癌组织中上皮细胞钙黏蛋白（E-cadherin）的表达情况,探讨E-cad与肿瘤侵袭、转移的关系。方法：收集临床确诊的存档鼻咽低分化鳞癌石蜡标本40例,鼻炎标本20例。将每个标本的4长切片分别进行HE染色、免疫组化PV二步法及阴性对照。HE确认病理类型,结合临床资病例料进行TNM分期,根据PV二步法染色结果检测E-cadherin的表达,所得结果用SPSSl7．0进行统计学检验。结果：E-cadherin在鼻咽癌组织对比中呈不同程度的降低（P=0．002）,与性别、年龄无明显相关,与T分期（P=0．023）、淋巴结转移（P=0．001）、TNM分期（P=0．000）显著相关。结论：1：E-cadherin在鼻咽癌组和对照组的表达有显著的差别,可能参与了鼻咽癌的发生发展。2：E-cadherin的表达与肿瘤的淋巴结转移和病理分期有相关性,但与年龄和性别无相关性。E-cadherin的表达缺失是肿瘤远处转移的重要指标。3：E-cadherin表达水平可能作为肿瘤侵袭,预测鼻咽癌颈部淋巴结隐匿性转移的潜在肿瘤标志物。     

EBV up-regulates cytochrome c through VDAC1 regulations and decreases the release of cytoplasmic Ca2+ in the NPC cell line

EBV (Epstein-Barr virus) is considered to be a major factor that causes NPC (nasopharyngeal carcinoma), which is one of the sneakiest cancers frequently occurring in Southeast Asia and Southern China. Apoptosis and pro-apoptotic signals have been studied for decades; however, few have extended the prevailing view of EBV to its impact on NPC in perspective of apoptosis. One of the important proteins named VDAC1 (voltage-dependent anion protein 1) on the mitochondrial outer membrane controls the pro-apoptotic signals in mammalian cells. The impact of EBV infection on VDAC1 and related apoptotic signals remains unclear. In order to study the VDAC1's role in EBV-infected NPC cells, we employ siRNA (small interfering RNA) inhibition to analyse the release of Ca2+ and Cyto c (cytochrome c) signals in the cytoplasm, as they are important pro-apoptotic signals. The results show a decrease of Ca2+ release and up-regulation of Cyto c with EBV infection. After siRNA transfection, the dysregulation of Cyto c is neutralized, which is evidence that the level of Cyto c release in virus-infected NPC cells is the as same as that of non-infected NPC cells. This result indicates that EBV infection changes the cytoplasmic level of Cyto c through regulating VDAC1. In summary, this study reports that EBV changes the release of Ca2+ and Cyto c in the cytoplasm of NPC cells, and that Cyto c changes are mediated by VDAC1 regulation.     

Epstein-Barr-virus-encoded LMP2A induces primary epithelial cell migration and invasion: possible role in nasopharyngeal carcinoma metastasis

Nonkeratinizing nasopharyngeal carcinomas (NPC) are >95% associated with the expression of the Epstein-Barr virus (EBV) LMP2A latent protein. However, the role of EBV, in particular, LMP2A, in tumor progression is not well understood. Using Affymetrix chips and a pattern-matching computational technique (neighborhood analysis), we show that the level of LMP2A expression in NPC biopsy samples correlates with that of a cellular protein, integrin-alpha-6 (ITGalpha6), that is associated with cellular migration in vitro and metastasis in vivo. We have recently developed a primary epithelial model from tonsil tissue to study EBV infection in epithelial cells. Here we report that LMP2A expression in primary tonsil epithelial cells causes them to become migratory and invasive, that ITGalpha6 RNA levels are up-regulated in epithelial cells expressing LMP2, and that ITGalpha6 protein levels are increased in the migrating cells. Blocking antibodies against ITGalpha6 abrogated LMP2-induced invasion through Matrigel by primary epithelial cells. Our results provide a link between LMP2A expression, ITGalpha6 expression, epithelial cell migration, and NPC metastasis and suggest that EBV infection may contribute to the high incidence of metastasis in NPC progression.     

天然免疫分子乳铁蛋白抑制鼻咽癌的发生发展

鼻咽癌是一种多基因遗传性肿瘤,其发病与遗传因素和环境因素密切相关,基因与环境因素间存在复杂的交互作用. 本课题组通过全基因组杂合性丢失扫描及比较基因组杂交,发现鼻咽癌中3号染色体短臂存在高频缺失,通过鼻咽癌家系连锁分析,发现染色体3p21区域为鼻咽癌易感基因区,随后通过表型克隆策略在该染色体区域分离鉴定了鼻咽癌候选易感/抑瘤基因LTF. LTF基因编码的乳铁蛋白是一种广泛分布于哺乳动物乳汁、鼻咽分泌物、泪液等分泌液中的天然免疫分子,在正常鼻咽部高表达而在鼻咽癌组织中表达显著下调,且与鼻咽癌的临床进展及侵袭转移密切相关. 病例-对照关联分析发现LTF基因中2个单核苷酸多态位点与鼻咽癌发病风险密切相关,且多态性改变可影响LTF基因的表达水平. 我们发现乳铁蛋白能与EB病毒在人B细胞表面的受体CD21结合,阻断EB病毒入侵宿主B细胞,并抑制EB病毒由B细胞向鼻咽上皮细胞的传递,在鼻咽上皮的癌变过程中起保护作用. 我们还发现LTF能通过MAPK和AKT等通路抑制鼻咽癌细胞的增殖和侵袭转移. 这些结果表明乳汁中的天然成份乳铁蛋白在鼻咽癌等EB病毒相关疾病的防治中具有重要的应用前景.     

Tissue inhibitor of matrix metalloproteinase-2 in nasopharyngeal carcinoma

By regulating matrix metalloproteinase (MMP) activity and controlling the breakdown of extracellular matrix components, tissue inhibitors of metalloproteinases (TIMPs) play an important role in the process of tumor invasion and metastasis. The present study was designed to clarify the role of TIMP-2 in nasopharyngeal carcinoma (NPC) patients and to evaluate its importance relative to clinicopathologic parameters. It was carried out in 30 patients with NPC and 20 controls. Tissue biopsies were studied and graded pathologically, and Western blot analysis was performed to assess TIMP-2 protein expression. Clinically, in accordance with TNM classification (T: tumor size, N: lymph node involvement, M: distant metastasis), 8 cases were diagnosed as stage II, 12 as stage III, and 10 cases as stage IV; however, pathologic typing with use of the World Health Organization (WHO) classification revealed the presence of 9 specimens of squamous cell carcinoma (WHO type 1), 6 cases of nonkeratinizing carcinoma (WHO type 2), and 15 cases of undifferentiated carcinoma (WHO type 3). The difference in percentage of TIMP-2 positivity between NPC patients (76.6%) and normal controls (30%) was statistically highly significant (P < .01). In addition, there was a significant positive correlation between TIMP-2 protein positivity and either the clinical staging or the histopathologic typing (P < .01) using Chi-square test (x(2)), suggesting that TIMP-2 can be used as a marker of the severity of NPC.Accordingly, we can assume that TIMP-2 may play a role in regional lymph node and/or distant metastasis and in progression of squamous cell carcinoma. Further studies are needed to investigate the role of TIMP-2 as a marker for tumor progression and to evaluate its potential value in the follow-up of patients.     

Epstein–Barr virus DNA quantification and follow-up in Tunisian nasopharyngeal carcinoma patients

The prognostic value of the Epstein–Barr virus (EBV) DNA load in sera of nasopharyngeal carcinoma (NPC) patients measured before any treatment, after treatment and before relapse was assessed. The real-time polymerase chain reaction was used to detect the viral load levels among 74 NPC subjects. Patients were followed up for a period going from 1 to 6 years (median 4 years). Before treatment, the EBV DNA load was correlated with lymph node involvement and advanced stages. After treatment, the viral load level declined significantly and patients presenting a viral load level lower than 1000 copies/ml showed a better overall survival (OS). Moreover, a significant result was found when the 6-year OS rates of patients having fewer or more than 15,000 copies/ml of viral load before relapse were compared. These results suggest that the EBV DNA load quantification after treatment may be a useful predictor of disease progression and survival.     

N,N′-Dinitrosopiperazine-Mediated AGR2 Is Involved in Metastasis of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) has a high metastatic character in the clinic, but its mechanism is not clear. As a carcinogen with organ specificity for the nasopharyngeal epithelium, N,N′-Dinitrosopiperazine (DNP) is involved in NPC metastasis. Herein, our data revealed that anterior gradient 2 (AGR2) was overexpressed in human NPC tissues, particularly in cervical lymph node metastatic NPC (LMNPC). High AGR2 expression was associated with NPC metastasis. Importantly, DNP induced AGR2 expression, and increased cell motility and invasion in the NPC cell line 6–10B. However, DNP-mediated cell motility and invasion was dramatically decreased when transfected with siRNA-AGR2. Further, AGR2 directly regulated cathepsin (CTS) B and D by binding them in vitro. These results indicate that DNP induces AGR2 expression, regulates CTSB and CTSD, increases cell motility and invasion, and promotes NPC tumor metastasis. Therefore, DNP-mediated AGR2 expression may be an important factor in prolific NPC metastasis.     

环状RNA参与鼻咽癌发生发展

环状RNA(circular RNA,circRNA)是一种具有共价闭环结构的非编码RNA(non-coding RNA,ncRNA),因其具有高稳定性、进化保守性和组织表达特异性的特点,越来越受到人们的关注。现有研究表明,circRNA参与恶性肿瘤等多种疾病的发生发展过程。鼻咽癌是一种起源于鼻咽上皮的恶性肿瘤,在中国华南和东南亚地区高发,发病与EB病毒（Epstein-Barr virus,EBV）感染密切相关。目前放疗和化疗是鼻咽癌治疗的主要手段,复发和远处转移是鼻咽癌患者死亡的主要原因。近年研究表明,circRNA作为基因表达调节因子,在鼻咽癌发生发展过程中发挥着重要的作用,影响着鼻咽癌的进展。本文主要综述了鼻咽癌中表达异常的circRNA,包括EB病毒编码的circRNA,在鼻咽癌发生发展中的研究现状,探讨了circRNA作为鼻咽癌患者治疗的潜在靶点以及诊断和预后标志物的可能性。     

Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments

Nasopharyngeal carcinoma (NPC) is a prevalent tumour in southern China and southeast Asia, particularly in the Cantonese population, where its incidence has remained high for decades. Recent studies have demonstrated that the aetiology of NPC is complex, involving multiple factors including genetic susceptibility, infection with the Epstein-Barr virus (EBV) and exposure to chemical carcinogens. During development of the disease, viral infection and multiple somatic genetic and epigenetic changes synergistically disrupt normal cell function, thus contributing to NPC pathogenesis. NPC is highly radiosensitive and chemosensitive, but treatment of patients with locoregionally advanced disease remains problematic. New biomarkers for NPC, including EBV DNA copy number or methylation of multiple tumour suppressor genes, which can be detected in serum and nasopharyngeal brushings, have been developed for the molecular diagnosis of this tumour. Meanwhile, new therapeutic strategies such as intensity-modulated radiation therapy and immuno- and epigenetic therapies might lead to more specific and effective treatments.     

Proteome profiling of Epstein-Barr virus infected nasopharyngeal carcinoma cell line: identification of potential biomarkers by comparative iTRAQ-coupled 2D LC/MS-MS analysis

Epstein-Barr virus (EBV) has been implicated in the development of nasopharyngeal carcinoma (NPC), a squamous cell carcinoma with high-occurrence in Southeast Asia and southern China. However, the underlying relationship of EBV and NPC squamous cell remains obscure. In this study, we employ a comparative iTRAQ-coupled 2D LC-MS/MS system to analyze the protein profile of NPC cell line upon EBV infection. Based on the proteome data and Western blot validation, 12 proteins were found to be significantly up-regulated and associated with signal transduction, cytoskeleton formation, metabolic pathways and DNA bindings. The interactions among NPC and EBV proteins were further analyzed and protein networks were established. Based on the functions of differentially expressed proteins, a metabolic pathway was proposed to elucidate their relationship in cytoskeleton formation, cell proliferation and apoptosis. Our results suggested a new proteome platform to analyze EBV's role in NPC squamous cell line. And these differentially expressed proteins may hold the promise as potential biomarkers for NPC diagnostics and treatment.