由 sufang 在 日, 08/03/2014 – 16:29 發表 Pre-published EBV Nasopharyngeal Carcinoma NPC
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1. Cheung, A. K., H. L. Lung, J. M. Ko, Y. Cheng, E. J. Stanbridge, E. R. Zabarovsky, J. M. Nicholls, D. Chua, S. W. Tsao, X. Y. Guan, and M. L. Lung. (2009) Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma. Proc Natl Acad Sci USA 106, 14478-83. (pdf 2348) Chromosome 14 allelic loss is common in nasopharyngeal carcinoma (NPC) and may reflect essential tumor suppressor gene loss in tumorigenesis. An intact chromosome 14 was transferred to an NPC cell line using a microcell-mediated chromosome transfer approach. Microcell hybrids (MCHs) containing intact exogenously transferred chromosome 14 were tumor suppressive in athymic mice, demonstrating that intact chromosome 14 NPC MCHs are able to suppress tumor growth in mice. Comparative analysis of these MCHs and their derived tumor segregants identified 4 commonly eliminated tumor-suppressive CRs. Here we provide functional evidence that a gene, Mirror-Image POLydactyly 1 (MIPOL1), which maps within a single 14q13.1-13.3 CR and that hitherto has been reported to be associated only with a developmental disorder, specifically suppresses in vivo tumor formation. MIPOL1 gene expression is down-regulated in all NPC cell lines and in approximately 63% of NPC tumors via promoter hypermethylation and allelic loss. SLC25A21 and FOXA1, 2 neighboring genes mapping to this region, did not show this frequent down-regulated gene expression or promoter hypermethylation, precluding possible global methylation effects and providing further evidence that MIPOL1 plays a unique role in NPC. The protein localizes mainly to the nucleus. Re-expression of MIPOL1 in the stable transfectants induces cell cycle arrest. MIPOL1 tumor suppression is related to up-regulation of the p21(WAF1/CIP1) and p27(KIP1) protein pathways. This study provides compelling evidence that chromosome 14 harbors tumor suppressor genes associated with NPC and that a candidate gene, MIPOL1, is associated with tumor development.
2. Hsu, W. L., J. Y. Chen, Y. C. Chien, M. Y. Liu, S. L. You, M. M. Hsu, C. S. Yang, and C. J. Chen. (2009) Independent effect of EBV and cigarette smoking on nasopharyngeal carcinoma: a 20-year follow-up study on 9,622 males without family history in Taiwan. Cancer Epidemiol Biomarkers Prev 18, 1218-26. (Hsu, Wan-Lun
Chen, Jen-Yang
Chien, Yin-Chu
Liu, Mei-Ying
You, San-Lin
Hsu, Mow-Ming
Yang, Czau-Siung
Chen, Chien-Jen
Research Support, Non-U.S. Gov’t
United States
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology
Cancer Epidemiol Biomarkers Prev. 2009 Apr;18(4):1218-26. Epub 2009 Mar 31.) This study aimed to assess independent effects of EBV and cigarette smoking on nasopharyngeal carcinoma, which have never been assessed in long-term follow-up studies. A cohort of 9,622 men was enrolled from 1984 to 1986. Blood samples collected at study entry were tested for antibodies against EBV antigens (anti-EBV) viral capsid antigen immunoglobulin A and DNase. The cigarette smoking habit was inquired through questionnaire interview. Newly developed nasopharyngeal carcinoma cases were ascertained through computerized linkage with national cancer registry profile. Cox’s proportional hazard regression analysis was used to estimate multivariate-adjusted hazard ratio with its 95% confidence interval (95% CI). During the follow-up of 173,706 person-years, 32 pathologically confirmed nasopharyngeal carcinoma cases were identified >1 year after recruitment. Increasing serum levels of anti-EBV viral capsid antigen immunoglobulin A and DNase were significantly associated with nasopharyngeal carcinoma risk in a dose-response relationship. The multivariate-adjusted hazard ratio (95% CI) of developing nasopharyngeal carcinoma for low and high antibody levels compared with seronegatives was 9.5 (2.2-40.1) and 21.4 (2.8-161.7), respectively, for anti-EBV viral capsid antigen immunoglobulin A (P < 0.001 for trend), and 1.6 (0.5-4.6) and 16.0 (5.4-47.1), respectively, for anti-EBV DNase (P < 0.001 for trend). The shorter the time interval between study entry and nasopharyngeal carcinoma diagnosis, the higher was the proportion of anti-EBV viral capsid antigen immunoglobulin A among nasopharyngeal carcinoma patients. The multivariate-adjusted hazard ratio (95% CI) was 3.0 (1.3-7.2) for > or =30 pack-years of cumulative cigarette smoking compared with <30 a="" and="" anti-ebv="" antigen="" are="" as="" capsid="" carcinoma="" cigarette="" dnase="" habit="" heavier="" heavy="" higher="" immunoglobulin="" independent="" long-term="" longer="" nasopharyngeal="" p="" pack-years="" predictors.="" reference.="" risk.="" risk="" smoking="" the="" viral="" was="">
Chen, Jen-Yang
Chien, Yin-Chu
Liu, Mei-Ying
You, San-Lin
Hsu, Mow-Ming
Yang, Czau-Siung
Chen, Chien-Jen
Research Support, Non-U.S. Gov’t
United States
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology
Cancer Epidemiol Biomarkers Prev. 2009 Apr;18(4):1218-26. Epub 2009 Mar 31.) This study aimed to assess independent effects of EBV and cigarette smoking on nasopharyngeal carcinoma, which have never been assessed in long-term follow-up studies. A cohort of 9,622 men was enrolled from 1984 to 1986. Blood samples collected at study entry were tested for antibodies against EBV antigens (anti-EBV) viral capsid antigen immunoglobulin A and DNase. The cigarette smoking habit was inquired through questionnaire interview. Newly developed nasopharyngeal carcinoma cases were ascertained through computerized linkage with national cancer registry profile. Cox’s proportional hazard regression analysis was used to estimate multivariate-adjusted hazard ratio with its 95% confidence interval (95% CI). During the follow-up of 173,706 person-years, 32 pathologically confirmed nasopharyngeal carcinoma cases were identified >1 year after recruitment. Increasing serum levels of anti-EBV viral capsid antigen immunoglobulin A and DNase were significantly associated with nasopharyngeal carcinoma risk in a dose-response relationship. The multivariate-adjusted hazard ratio (95% CI) of developing nasopharyngeal carcinoma for low and high antibody levels compared with seronegatives was 9.5 (2.2-40.1) and 21.4 (2.8-161.7), respectively, for anti-EBV viral capsid antigen immunoglobulin A (P < 0.001 for trend), and 1.6 (0.5-4.6) and 16.0 (5.4-47.1), respectively, for anti-EBV DNase (P < 0.001 for trend). The shorter the time interval between study entry and nasopharyngeal carcinoma diagnosis, the higher was the proportion of anti-EBV viral capsid antigen immunoglobulin A among nasopharyngeal carcinoma patients. The multivariate-adjusted hazard ratio (95% CI) was 3.0 (1.3-7.2) for > or =30 pack-years of cumulative cigarette smoking compared with <30 a="" and="" anti-ebv="" antigen="" are="" as="" capsid="" carcinoma="" cigarette="" dnase="" habit="" heavier="" heavy="" higher="" immunoglobulin="" independent="" long-term="" longer="" nasopharyngeal="" p="" pack-years="" predictors.="" reference.="" risk.="" risk="" smoking="" the="" viral="" was="">
3. Tse, K. P., W. H. Su, K. P. Chang, N. M. Tsang, C. J. Yu, P. Tang, L. C. See, C. Hsueh, M. L. Yang, S. P. Hao, H. Y. Li, M. H. Wang, L. P. Liao, L. C. Chen, S. R. Lin, T. J. Jorgensen, Y. S. Chang, and Y. Y. Shugart. (2009) Genome-wide association study reveals multiple nasopharyngeal carcinoma-associated loci within the HLA region at chromosome 6p21.3. Am J Hum Genet 85, 194-203. (pdf 2613. (1) GWAS in 277 NPC patiwnts and 285 healthy controls. (2) Figure 3 EBER In situ staining 強而美麗!) Nasopharyngeal carcinoma (NPC) is a multifactorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. To identify the common genetic variants linked to NPC susceptibility, we conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3. Associations were replicated in two independent sets of case-control samples. Two of the most significant SNPs (rs2517713 and rs2975042; p(combined) = 3.9 x 10(-20) and 1.6 x 10(-19), respectively) were located in the HLA-A gene. Moreover, we detected significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 (GABBR1) (rs29232; p(combined) = 8.97 x 10(-17)) and HLA-F (rs3129055 and rs9258122; p(combined) = 7.36 x 10(-11) and 3.33 x 10(-10), respectively). Notably, the association of rs29232 remained significant (residual p < 5 x 10(-4)) after adjustment for age, gender, and HLA-related SNPs. Furthermore, higher GABA(B) receptor 1 expression levels can be found in the tumor cells in comparison to the adjacent epithelial cells (p < 0.001) in NPC biopsies, implying a biological role of GABBR1 in NPC carcinogenesis. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Although some of these relationships may be attributed to linkage disequilibrium between the loci, the findings clearly provide a fresh direction for the study of NPC development.
4. Alajez, N. M., W. Shi, A. B. Hui, J. Bruce, M. Lenarduzzi, E. Ito, S. Yue, B. O’Sullivan, and F. F. Liu. (2010) Enhancer of Zeste homolog 2 (EZH2) is overexpressed in recurrent nasopharyngeal carcinoma and is regulated by miR-26a, miR-101, and miR-98. Cell Death Dis 1, e85. (SFL notes: (1) Same group (at Canada) from Ref 38 in Chen and Zhou) There is increasing evidence supporting the role of members of the polycomb group (PcG) gene family in tumor development and progression. However, their precise role in tumorigenesis and mechanisms of their regulation remain to be elucidated. Using nasopharyngeal carcinoma (NPC) as a disease model, a comprehensive analysis was undertaken on the clinical significance of EZH2 expression, identification of the cellular processes regulated by EZH2, and the mechanisms of its deregulated expression. Herein, we report EZH2 as being associated with a higher risk of relapse in NPC patients (P = 0.002). Genome-wide microarray and bioinformatics identified several vital cellular processes (such as differentiation, development, and apoptosis) to be regulated by EZH2, corroborated by in vitro lethality, and delayed tumor formation in vivo upon EZH2 depletion. The combination of global microRNA (miR) profiling in primary NPC specimens, and in silico analyses provided several candidate miRs that could regulate EZH2. Using a luciferase-based assay, miR-26a, miR-101, and miR-98 were validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in NPC biology.
5. Bei, J. X., Y. Li, W. H. Jia, B. J. Feng, G. Zhou, L. Z. Chen, Q. S. Feng, H. Q. Low, H. Zhang, F. He, E. S. Tai, T. Kang, E. T. Liu, J. Liu, and Y. X. Zeng. (2010) A genome-wide association study of nasopharyngeal carcinoma identifies three new susceptibility loci. Nat Genet 42, 599-603. (pdf 2612. 曾益興 GWAS studies. (1) 3507 cases and 3063 controls of the southern Chinese decent feom Guangdong and Guanxi. (2) 之前長庚張玉生老師 (pdf 2613) 他們identifiy chromosome 6p21.3 HLA-A (*1101), GABBR1 and HLA-F 有得到這篇作者的confirm. 另外這篇則新加了 13q12 (TNFRSF19), 3q26 (MDS1-EVI1), and 9p21 (CDKN2A-CDKN2B).) To identify genetic susceptibility loci for nasopharyngeal carcinoma (NPC), a genome-wide association study was performed using 464,328 autosomal SNPs in 1,583 NPC affected individuals (cases) and 1,894 controls of southern Chinese descent. The top 49 SNPs from the genome-wide association study were genotyped in 3,507 cases and 3,063 controls of southern Chinese descent from Guangdong and Guangxi. The seven supportive SNPs were further confirmed by transmission disequilibrium test analysis in 279 trios from Guangdong. We identified three new susceptibility loci, TNFRSF19 on 13q12 (rs9510787, Pcombined=1.53×10(-9), odds ratio (OR)=1.20), MDS1-EVI1 on 3q26 (rs6774494, Pcombined=1.34×10(-8), OR=0.84) and the CDKN2A-CDKN2B gene cluster on 9p21 (rs1412829, Pcombined=4.84×10(-7), OR=0.78). Furthermore, we confirmed the role of HLA by revealing independent associations at rs2860580 (Pcombined=4.88×10(-67), OR=0.58), rs2894207 (Pcombined=3.42×10(-33), OR=0.61) and rs28421666 (Pcombined=2.49×10(-18), OR=0.67). Our findings provide new insights into the pathogenesis of NPC by highlighting the involvement of pathways related to TNFRSF19 and MDS1-EVI1 in addition to HLA molecules.
6. Braz-Silva, P. H., S. Vitale, C. Butori, N. Guevara, J. Santini, M. Magalhaes, P. Hofman, and A. Doglio. (2010) Specific infiltration of langerin-positive dendritic cells in EBV-infected tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma. Int J Cancer (pdf_.) We report here the existence of a novel subset of langerin (CD207)-positive, immature dendritic cells (DCs) (CD83(neg)) abundantly infiltrating Epstein Barr virus (EBV)-infected areas in tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma. These CD207(+) DCs differ from conventional epidermal Langerhans cells in their lack of CD1a and CCR6 and their unusual tissue localization. CD207(+) DC infiltration strongly correlates with EBV infection because it was neither detected in EBV negative specimens nor in tissues infected with other human viruses. These immature DCs might represent good candidates for induction of the EBV-specific immune response.
7. Mousavi, S. M., J. Sundquist, and K. Hemminki. (2010) Nasopharyngeal and hypopharyngeal carcinoma risk among immigrants in Sweden. Int J Cancer (Journal article) Environmental exposures, particularly infection with Epstein-Barr virus (EBV) and tobacco are known risk factors for oral cancer. Studies in migrants may provide valuable insight into the environmental and genetic etiology of cancer. We wanted to define nasopharyngeal and hypopharyngeal carcinoma among immigrants in Sweden.The nationwide Swedish Family-Cancer Database (FCD) was used to calculate standardized incidence ratios (SIRs) for nasopharyngeal and hypopharyngeal carcinomas among the first-generation immigrants compared to the native Swedes.The FCD included 1969 and 691 cases of nasopharyngeal and hypopharyngeal carcinoma in the male and female Swedes, and 178 and 65 cases in immigrants, respectively. The median age at diagnosis (years) was 63 among Swedes and 55 among immigrants. The risk of nasopharyngeal carcinoma was significantly higher in male (SIR= 35.6) and female (24.6) Southeast Asians, male (12.4) and female (34.7) North Africans, male (4.9) and female (10.9) Asian Arabs, and some other male Asians immigrants (6.2 to 6.7). Among immigrants from European countries, only the men from former Yugoslavian showed an elevated risk (2.7). Hypopharyngeal carcinoma risk was only increased among the male immigrants from the Indian Subcontinent (5.4).Early life infection with EBV in countries of origin and probably a minor contribution by tobacco smoking may be the main environmental exposures influencing nasopharyngeal carcinoma risks among immigrants to Sweden. The high rates of hypopharyngeal carcinoma among Indian immigrants may point to a continued using of smokeless tobacco. (c) 2010 UICC.
8. Tsang, C. M., G. Zhang, E. Seto, K. Takada, W. Deng, Y. L. Yip, C. Man, P. M. Hau, H. Chen, Y. Cao, K. W. Lo, J. M. Middeldorp, A. L. Cheung, and S. W. Tsao. (2010) Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization. Int J Cancer 127, 1570-83. (pdf 2579. transient lytic, abortive lytic) Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-beta effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBV-encoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-alpha. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development.
9. Liu, P., X. Fang, Z. Feng, Y. M. Guo, R. J. Peng, T. Liu, Z. Huang, Y. Feng, X. Sun, Z. Xiong, X. Guo, S. S. Pang, B. Wang, X. Lv, F. T. Feng, D. J. Li, L. Z. Chen, Q. S. Feng, W. L. Huang, M. S. Zeng, J. X. Bei, Y. Zhang, and Y. X. Zeng. (2011) Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology. J Virol 85, 11291-9. (Pdf__ 強! I respect you.) Epstein-Barr virus (EBV)-encoded molecules have been detected in the tumor tissues of several cancers, including nasopharyngeal carcinoma (NPC), suggesting that EBV plays an important role in tumorigenesis. However, the nature of EBV with respect to genome width in vivo and whether EBV undergoes clonal expansion in the tumor tissues are still poorly understood. In this study, next-generation sequencing (NGS) was used to sequence DNA extracted directly from the tumor tissue of a patient with NPC. Apart from the human sequences, a clinically isolated EBV genome 164.7 kb in size was successfully assembled and named GD2 (GenBank accession number HQ020558). Sequence and phylogenetic analyses showed that GD2 was closely related to GD1, a previously assembled variant derived from a patient with NPC. GD2 contains the most prevalent EBV variants reported in Cantonese patients with NPC, suggesting that it might be the prevalent strain in this population. Furthermore, GD2 could be grouped into a single subtype according to common classification criteria and contains only 6 heterozygous point mutations, suggesting the monoclonal expansion of GD2 in NPC. This study represents the first genome-wide analysis of a clinical isolate of EBV directly extracted from NPC tissue. Our study reveals that NGS allows the characterization of genome-wide variations of EBV in clinical tumors and provides evidence of monoclonal expansion of EBV in vivo. The pipeline could also be applied to the study of other pathogen-related malignancies. With additional NGS studies of NPC, it might be possible to uncover the potential causative EBV variant involved in NPC.
10. Hsu, W. L., K. P. Tse, S. Liang, Y. C. Chien, W. H. Su, K. J. Yu, Y. J. Cheng, N. M. Tsang, M. M. Hsu, K. P. Chang, I. H. Chen, T. I. Chen, C. S. Yang, A. M. Goldstein, C. J. Chen, Y. S. Chang, and A. Hildesheim. (2012) Evaluation of Human Leukocyte Antigen-A (HLA-A), Other Non-HLA Markers on Chromosome 6p21 and Risk of Nasopharyngeal Carcinoma. PLoS One 7, e42767. (pdf__
Hsu, Wan-Lun
Tse, Ka-Po
Chen, Chien-Jen
Chang, Yu-Sun
Hildesheim, Allan
United States
PloS one
PLoS One. 2012;7(8):e42767. Epub 2012 Aug 7.) BACKGROUND: The association between human leukocyte antigen (HLA) genes (located in the Major Histocompatibility Complex [MHC] region of chromosome 6p21) and NPC has been known for some time. Recently, two genome-wide association studies (GWAS) conducted in Taiwan and China confirmed that the strongest evidence for NPC association was mapped to the MHC region. It is still unclear, however, whether these findings reflect direct associations with Human Leukocyte Antigen (HLA) genes and/or to other genes in this gene-rich region. METHODS: To better understand genetic associations for NPC within the MHC region of chromosome 6, we conducted an evaluation that pooled two previously conducted NPC case-control studies in Taiwan (N = 591 cases and N = 521 controls). PCR-based genotyping was performed for 12 significant SNPs identified within 6p21 in the Taiwan NPC GWAS and for the HLA-A gene (exons 2 and 3). FINDINGS: After confirming homogeneity between the two studies, pooled odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression. We found that HLA-A (p-trend = 0.0006) and rs29232 (within the GABBR1 gene; p-trend = 0.005) were independent risk factors for NPC after adjustment for age, gender, study and each other. NPC risk was highest among individuals who were homozygous for the HLA-A*0207 risk allele and carriers of the rs29232 risk allele (A). CONCLUSION: Our study suggests that most of the SNPs significantly associated with NPC from GWAS reflect previously identified HLA-A associations. An independent effect of rs29232 (GABBR1), however, remained, suggesting that additional genes within this region might be associated with NPC risk.
Hsu, Wan-Lun
Tse, Ka-Po
Chen, Chien-Jen
Chang, Yu-Sun
Hildesheim, Allan
United States
PloS one
PLoS One. 2012;7(8):e42767. Epub 2012 Aug 7.) BACKGROUND: The association between human leukocyte antigen (HLA) genes (located in the Major Histocompatibility Complex [MHC] region of chromosome 6p21) and NPC has been known for some time. Recently, two genome-wide association studies (GWAS) conducted in Taiwan and China confirmed that the strongest evidence for NPC association was mapped to the MHC region. It is still unclear, however, whether these findings reflect direct associations with Human Leukocyte Antigen (HLA) genes and/or to other genes in this gene-rich region. METHODS: To better understand genetic associations for NPC within the MHC region of chromosome 6, we conducted an evaluation that pooled two previously conducted NPC case-control studies in Taiwan (N = 591 cases and N = 521 controls). PCR-based genotyping was performed for 12 significant SNPs identified within 6p21 in the Taiwan NPC GWAS and for the HLA-A gene (exons 2 and 3). FINDINGS: After confirming homogeneity between the two studies, pooled odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression. We found that HLA-A (p-trend = 0.0006) and rs29232 (within the GABBR1 gene; p-trend = 0.005) were independent risk factors for NPC after adjustment for age, gender, study and each other. NPC risk was highest among individuals who were homozygous for the HLA-A*0207 risk allele and carriers of the rs29232 risk allele (A). CONCLUSION: Our study suggests that most of the SNPs significantly associated with NPC from GWAS reflect previously identified HLA-A associations. An independent effect of rs29232 (GABBR1), however, remained, suggesting that additional genes within this region might be associated with NPC risk.
11. Hu, C., W. Wei, X. Chen, C. B. Woodman, Y. Yao, J. M. Nicholls, I. Joab, S. K. Sihota, J. Y. Shao, K. D. Derkaoui, A. Amari, S. L. Maloney, A. I. Bell, P. G. Murray, C. W. Dawson, L. S. Young, and J. R. Arrand. (2012) A global view of the oncogenic landscape in nasopharyngeal carcinoma: an integrated analysis at the genetic and expression levels. PLoS One 7, e41055. (pdf__) Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.
12. Kwok, H., A. H. Tong, C. H. Lin, S. Lok, P. J. Farrell, D. L. Kwong, and A. K. Chiang. (2012) Genomic sequencing and comparative analysis of epstein-barr virus genome isolated from primary nasopharyngeal carcinoma biopsy. PLoS One 7, e36939. (no pdf. (Has P Farrel!!) ) Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt’s lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8(+) T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.
13. Lun, S. W., S. T. Cheung, P. F. Cheung, K. F. To, J. K. Woo, K. W. Choy, C. Chow, C. C. Cheung, G. T. Chung, A. S. Cheng, C. W. Ko, S. W. Tsao, P. Busson, M. H. Ng, and K. W. Lo. (2012) CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma. PLoS One 7, e52426. (pdf not yet Lo, Kwok-Wai) Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.
14. Man Tsang, C., Y. L. Yip, K. W. Lo, W. Deng, K. F. To, P. Man Hau, V. M. Lau, K. Takada, V. W. Lui, M. L. Lung, H. Chen, M. Zeng, J. M. Middeldorp, A. L. Cheung, and S. W. Tsao. (2012) Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells. Proc Natl Acad Sci USA (pdf 3562 in BB2012Dec. Tso SW, 2012-12. Rta/CTCF/Senescence related.) Undifferentiated nasopharyngeal carcinomas (NPCs) are commonly present with latent EBV infection. However, events regulating EBV infection at early stages of the disease and the role of EBV in disease pathogenesis are largely undefined. Genetic alterations leading to activation of cyclin D1 signaling in premalignant nasopharyngeal epithelial (NPE) cells have been postulated to predispose cells to EBV infection. We previously reported that loss of p16, a negative regulator of cyclin D1 signaling, is a frequent feature of NPC tumors. Here, we report that early premalignant lesions of nasopharyngeal epithelium overexpress cyclin D1. Furthermore, overexpression of cyclin D1 is closely associated with EBV infection. Therefore we investigated the potential role of cyclin D1 overexpression in dysplastic NPE cells in vitro. In human telomerase reverse transcriptase-immortalized NPE cells, overexpression of cyclin D1 or a p16-resistant form of CDK4 (CDK4(R24C)) suppressed differentiation. This suppression may have implications for the close association of EBV infection with undifferentiated NPC. In these in vitro models, we found that cellular growth arrest and senescence occurred in EBV-infected cell populations immediately after infection. Nevertheless, overexpression of cyclin D1 or a p16-resistant form of CDK4 or knockdown of p16 in the human telomerase reverse transcriptase-immortalized NPE cell lines could counteract the EBV-induced growth arrest and senescence. We conclude that dysregulated expression of cyclin D1 in NPE cells may contribute to NPC pathogenesis by enabling persistent infection of EBV.
15. Tang, M., J. A. Lautenberger, X. Gao, E. Sezgin, S. L. Hendrickson, J. L. Troyer, V. A. David, L. Guan, C. E. McIntosh, X. Guo, Y. Zheng, J. Liao, H. Deng, M. Malasky, B. Kessing, C. A. Winkler, M. Carrington, G. De The, Y. Zeng, and S. J. O’Brien. (2012) The Principal Genetic Determinants for Nasopharyngeal Carcinoma in China Involve the HLA Class I Antigen Recognition Groove. PLoS Genet 8, e1003103. (no pdf yet. NPC HLA on PLoS Pathogens) Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA -B, and HLA -C class I genes (P(HLA-A-aa-site-62) = 7.4×10(-29); P (HLA-B-aa-site-116) = 6.5×10(-19); P (HLA-C-aa-site-156) = 6.8×10(-8) respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China.
16. Toh, H. C., T. C. Ha, and J. Wee. (2012) Personalised medicine in nasopharyngeal cancer. The lancet oncology 13, 568-9. (pdf__.) The main principle of traditional Chinese medicine—which began more than 4000 years ago and is summarised in The Yellow Emperor’s Canon of Medicine—is the notion of syndrome differentiation and treatment. This notion aims to analyse, induce, synthesise, judge, and summarise the clinical data of symptoms and signs collected with four diagnostic methods (inspection, listening and smelling, inquiry, and pulse reading and palpation) into a diagnosis of one syndrome. Therapeutic strategies are then decided according to the result of syndrome differentiation. Thus, although people living hundreds or thousands of years ago did not have access to next-generation sequencers, they were nevertheless very astute observers of nature and human beings.
A study1 published in 2010 showed that different radiological signs of nasopharyngeal carcinoma (eg, cervical lymph node metastases, skull-base erosions, and radiosensitivity) could be correlated with different syndrome types from traditional Chinese medicine. In another study2 of 706 individuals, Chen and colleagues showed a possible genetic basis for the classification of physical constitution in traditional Chinese medicine. Therefore, syndrome differentiation as per traditional Chinese medicine might provide a subtle phenotypic subclassification within an allopathic disease diagnosis, suggesting an underlying genotypic difference.
Fast forward to modern allopathic medicine and the TNM system, which has been in existence since the 1940s, and use of classification from anatomical prognostic factors has withstood the test of time. Such anatomical assessment remains the basis of personalised medicine.
In nasopharyngeal carcinoma, a group from Hong Kong successfully tailored therapy by reducing the radiotherapy dose to the pituitary fossa and neck for patients without cancer involvement of those structures. This group’s next attempt at personalised medicine was confirmation of the usefulness of addition of concurrent chemotherapy to radiotherapy in two trials. The first trial (NCT00563927) tested the addition of chemotherapy for patients at highest risk of development of distant metastases and the second (NCT00563862) had a component of accelerated radiation in a subset of patients at increased risk for local failure. The same group is presently assessing the use of Epstein-Barr virus (EBV) DNA levels after radiotherapy to identify patients who would benefit from additional adjuvant treatment (NCT00370890).
Use of molecular signatures has proven to be important for the prediction of outcomes of many cancers. A group from Guangzhou (Guangdong, China) led an international consortium3 to identify an eight signature classifier for prediction of survival in nasopharyngeal carcinoma. MicroRNAs (miRNAs) make up a large class of small non-protein-coding RNAs that are very influential in gene regulation and cellular function. Complex interactions between miRNAs, and their dysregulation, can amplify diverse pathogenic processes that contribute to initiation of tumour growth, proliferation, and metastases. miRNAs have prognostic significance in various haematological and solid tumours. In The Lancet Oncology, Liu and colleagues4 report a distinct miRNA profile derived from nasopharyngeal carcinoma tumour tissue, and a five miRNA signature that independently predicts disease-free and overall survival and correlates with TNM staging. Moreover, in combination with TNM staging this miRNA signature improves the prognostic value versus TNM alone in terms of survival. This signature-based scoring could improve oncologists’ ability to identify patients who will likely have poorer outcomes and therefore plan improved therapeutic interventions. Liu and colleagues should be commended for their retrospective analysis of systematically annotated specimens, because biopsy material is usually small in nasopharyngeal carcinoma compared with other cancers in which surgery is definitively required. Their study is also the first comprehensive assessment of miRNAs as prognostic biomarkers in this cancer. Such a study potentially allows for increased probing of the biological mechanisms underpinning the role of miRNA in the oncogenesis of this EBV-transformed cancer. Ideally, investigators will eventually be able to replicate and validate this tumour tissue 5-miRNA signature in patients’ serum, which is easier to access and contains miRNA that is as stable as that of tissue. Another report has suggested that serum EBV miRNA of patients with nasopharyngeal carcinoma correlates positively with cellular copy numbers of EBV miRNAs.5 The same group reported that EBV miRNAs were able to inhibit tumour suppressor genes such as PTEN and deregulate pathways including Wnt signalling.
As pointed out by Liu and colleagues, elucidation of the intricate network and roles of miRNA in development of nasopharyngeal carcinoma could eventually allow for specific in-vivo therapeutic targeting. Chemotherapy and radiotherapy form the cornerstone of treatment of nasopharyngeal carcinoma. A few molecularly targeting drugs are emerging as clinically active against advanced nasopharyngeal carcinoma. [6] and [7] Use of miRNA signatures in clinical decision-making for prediction of treatment efficacy and increasingly tailored therapy will be the next step forward, especially for metastatic nasopharyngeal carcinoma for which no phase 3 therapeutic clinical trials have been done so far.
17. Adham, M., A. E. Greijer, S. A. Verkuijlen, H. Juwana, S. Fleig, L. Rachmadi, O. Malik, A. N. Kurniawan, A. Roezin, S. Gondhowiardjo, D. Atmakusumah, S. J. Stevens, B. Hermani, I. B. Tan, and J. M. Middeldorp. (2013) Epstein-Barr virus DNA load in nasopharyngeal brushings and whole blood in nasopharyngeal carcinoma patients before and after treatment. Clin Cancer Res 19, 2175-86. (pdf 3860.) PURPOSE: Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) and highly prevalent in Indonesia. EBV-DNA load can be used for early diagnosis and may have prognostic value. In this study, EBV-DNA load was evaluated in minimal invasive nasopharyngeal (NP) brushings and whole blood for initial diagnosis and therapy assessment against the standard-of-care diagnosis by biopsy with EBV-RISH and standard EBV-IgA serology. EXPERIMENTAL DESIGN: NP brushings and blood samples were collected from 289 consecutive ENT patients suspected of NPCs and 53 local healthy controls. EBV-DNA load was quantified by real-time PCR and serology by peptide-based EBV-IgA ELISA. Tissue biopsies were examined by routine histochemistry and by EBER RNA in situ hybridization. RESULTS: Repeated NP brushing was well tolerated by patients and revealed high viral load in the 228 NPC cases at diagnosis than 61 non-NPC cancer cases and healthy controls (P < 0.001). The diagnostic value of EBV-DNA load in blood and EBV-IgA serology was inferior to the NP brush results. The level of EBV-DNA load in brushes of patients with NPC was not related to T, N, or M stage, whereas elevated EBV-DNA load in blood correlated with N and M stage. EBV-DNA levels in brushings and whole blood showed a significant reduction at 2 months after treatment (P = 0.001 and P = 0.005, respectively), which was not reflected in EBV-IgA serology. CONCLUSIONS: NP brush sampling combined with EBV-DNA load analysis is a minimal invasive and well-tolerated diagnostic procedure, suited for initial diagnosis and follow-up monitoring of NPCs.
18. Chung, G. T., R. W. Lung, A. B. Hui, K. Y. Yip, J. K. Woo, C. Chow, C. Y. Tong, S. D. Lee, J. W. Yuen, S. W. Lun, K. K. Tso, N. Wong, S. W. Tsao, T. T. Yip, P. Busson, H. Kim, J. S. Seo, B. O’Sullivan, F. F. Liu, K. F. To, and K. W. Lo. (2013) Identification of a recurrent transforming UBR5-ZNF423 fusion gene in EBV-associated nasopharyngeal carcinoma. J Pathol 231, 158-67. (pdf 4127.) Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is prevalent in southern China, south-east Asia and northern Africa. The development and stepwise progression of NPC involves accumulation of multiple gross genetic changes during the clonal expansion of Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cell population. Here, using paired-end whole-transcriptome sequencing, we discovered a number of chimeric fusion transcripts in a panel of EBV-positive tumour lines. Among these transcripts, a novel fusion of ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and zinc finger protein 423 (ZNF423) on 16q12.1, identified from the NPC cell line C666-1, was recurrently detected in 12/144 (8.3%) of primary tumours. The fusion gene contains exon 1 of UBR5 and exons 7-9 of ZNF423 and produces a 94 amino acid chimeric protein including the original C-terminal EBF binding domain (ZF29-30) of ZNF423. Notably, the growth of NPC cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by fusion-specific siRNA significantly inhibited the cell proliferation and colony-forming ability of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 fibroblasts. Constitutive expression of UBR5-ZNF423 in NIH3T3 fibroblasts significantly enhanced its anchorage-independent growth in soft agar and induced tumour formation in a nude mouse model. These findings suggest that expression of UBR5-ZNF423 protein might contribute to the transformation of a subset of NPCs, possibly by altering the activity of EBFs (early B cell factors). Identification of the oncogenic UBR5-ZNF423 provides new potential opportunities for therapeutic intervention in NPC.
19. Fogg, M., J. R. Murphy, J. Lorch, M. Posner, and F. Wang. (2013) Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein-Barr virus associated nasopharyngeal carcinoma. Virology 441, 107-13. (no pdf yet. T-Reg in NOC treatment.) Epstein-Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, as well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients.
20. Hui, E. P., G. S. Taylor, H. Jia, B. B. Ma, S. L. Chan, R. Ho, W. L. Wong, S. Wilson, B. F. Johnson, C. Edwards, D. D. Stocken, A. B. Rickinson, N. M. Steven, and A. T. Chan. (2013) Phase I trial of recombinant modified vaccinia ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients. Cancer research 73, 1676-88. (no pdf yet. NPC Vaccine (EBNA1 and LMP1)) Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 x 10(7) and 5 x 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-gamma ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.
21. Le, Q. T., Q. Zhang, H. Cao, A. J. Cheng, B. A. Pinsky, R. L. Hong, J. T. Chang, C. W. Wang, K. C. Tsao, Y. D. Lo, N. Lee, K. K. Ang, A. T. Chan, and K. C. Chan. (2013) An international collaboration to harmonize the quantitative plasma Epstein-Barr virus DNA assay for future biomarker-guided trials in nasopharyngeal carcinoma. Clin Cancer Res 19, 2208-15. (pdf 3823.) PURPOSE: Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation. EXPERIMENTAL DESIGN: Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples. RESULTS: The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39-0.78], 0.70 (0.50-0.83), and 0.59 (0.35-0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5-0.95), 0.95 (0.83-0.99) and 0.96 (0.86-0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA. CONCLUSIONS: Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability.
22. Li, H. P., C. C. Peng, I. C. Chung, M. Y. Huang, S. T. Huang, C. C. Chen, K. P. Chang, C. L. Hsu, and Y. S. Chang. (2013) Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in Nasopharyngeal carcinoma. Oncotarget (Li, Hsin-Pai
Peng, Chen-Ching
Chung, I-Che
Huang, Mei-Yuan
Huang, Shao-Tung
Chen, Chia-Chun
Chang, Kai-Ping
Hsu, Cheng-Lung
Chang, Yu-Sun
Oncotarget. 2013 Nov 4.) Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5’Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptionalrepression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 wascorrelated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.
Peng, Chen-Ching
Chung, I-Che
Huang, Mei-Yuan
Huang, Shao-Tung
Chen, Chia-Chun
Chang, Kai-Ping
Hsu, Cheng-Lung
Chang, Yu-Sun
Oncotarget. 2013 Nov 4.) Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5’Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptionalrepression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 wascorrelated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.
23. Liao, L. J., H. W. Chou, C. T. Wang, C. S. Chung, and M. S. Lai. (2013) The impact of second primary malignancies on head and neck cancer survivors: a nationwide cohort study. PLoS One 8, e62116. (賴美淑. HNSCC second primary.) BACKGROUND: Head and neck cancer (HNC) is associated with a high rate of developing second primary malignancies(SPMs). But the impact on survival remains poorly understood before. Therefore, we want to estimate the impact of SPMs on HNC survivors. METHODS AND FINDINGS: Between 1986 and 2008, a total of 9,996 SPMs were recorded for 93,891 patients with an initial diagnosis of HNC by the Taiwan Cancer Registry. Patients were followed with national death registry database to 2011.Using the Kaplan-Meier method, a time-dependent covariate was employed to compare the survival rates between patients with and without SPMs. A Cox proportional hazards model that treated age and sex as confounders was used to examine the hazard ratios of SPMs. The relative survival rates were calculated using age- and sex-specific life tables for the population. Parametric mixture cure fraction models were then employed to estimate the percentage of cancer survivors who would be cured. Use of the Kaplan-Meier method showed that the crude survival rates differed significantly for patients with and patients without SPMs (log-rank test <0 .01="" 11="" 2.28="" 2.34="" 2.40="" 2.53to="" 2.59="" 2.65="" 39="" a="" analysis="" and="" as="" at="" cancer.="" cancer="" carcinoma="" ci="" clinical="" conclusions:="" consider="" cox="" cure="" esophageal="" for="" found="" had="" have="" hazards="" healthcare="" high="" highest="" influence="" lung="" multivariate="" must="" nasopharyngeal="" of="" on="" or="" p="" patients="" primary="" prognosis="" proportional="" providers="" rate="" rates="" regression="" results="" second="" significant="" spms.="" spms="" strongly="" such="" survival="" suspicion="" the="" these="" to="" univariate="" was="" where="" with="" worse="" worst="">
24. Lin, P., and Z. Huang. (2013) Correlation analysis connects cancer subtypes. PLoS One 8, e69747. (pdf 3747 in BB2013Sep, worth checking out subtypes for lung SCC and NPC. ) We provided a cross-tissue comparative analysis of between-subtype molecular commonality for ovarian cancer, breast cancer, hepatocellular carcinoma, glioma, lung squamous carcinoma and nasopharyngeal carcinoma. Our analysis showed that molecular subtypes with similar phenotype or similar clinical outcome could be correlated by their transcriptional profile and pathway profile. Pathway dysregulation across multiple cancer subtypes was also revealed by Gene Set Enrichment Analysis. Dysregulation of ‘complement and coagulation cascades’ was observed in a total of eleven subtypes across five tissues, implicating that the role of this process in personalized immune-based therapy may be worth further exploring.
25. Liu, Y. T., Y. Y. Fan, C. H. Xu, X. L. Lin, Y. K. Lu, X. L. Zhang, C. X. Zhang, and Y. M. Chen. (2013) Habitual consumption of soy products and risk of nasopharyngeal carcinoma in chinese adults: a case-control study. PLoS One 8, e77822. (no pdf yet.) BACKGROUND AND OBJECTIVES: Many studies have shown a negative association between the consumption of soy products and the risk of some cancers, but little is known about the effect of soy consumption on nasopharyngeal carcinoma. We assessed the association between the consumption of soy products on nasopharyngeal carcinoma risk in Chinese individuals. METHODS: This case-control study included 600 (448 males and 152 females) incident cases of nasopharyngeal carcinoma, and an equal number of controls, matched according to gender, age (+/- 3 y) and household type to the nasopharyngeal carcinoma cases. All subjects were recruited from hospitals in Guangzhou, China. A face-to-face interview was conducted with each study individual to collect general information and habitual dietary intake using a 78-item quantitative food-frequency questionnaire. Odds ratios and their 95% confidence intervals were estimated using conditional logistic regression analyses. RESULTS: The median intakes of soy foods (in protein) were 0.5/0.5, 1.4/1.7, 2.7/3.3 and 6.1/7.7 (male/female) g/d in the quartiles 1 to 4. Both univariate and multivariate analyses showed no significant association between the consumption of soy proteins or soy isoflavones and the risk of nasopharyngeal carcinoma. The adjusted odds ratios (95% confidence intervals) between extreme quartiles were 0.97 (0.66-1.45) for soy proteins and 0.97 (0.66-1.42) for total isoflavones. Null associations were also observed between intake of the individual isoflavones daidzein, genistein and glycitein and NPC risk, with adjusted odds ratios for the extreme quartiles ranging between 0.73 and 1.23. CONCLUSION: Habitual consumption of soy products had no significant effect on the risk of nasopharyngeal carcinoma in Chinese adults with a relatively low intake.
26. Luftig, M. (2013) Heavy LIFting: tumor promotion and radioresistance in NPC. The Journal of clinical investigation 123, 1-3. (Luftig, Micah
J Clin Invest. 2013 Nov 25:1-3. doi: 10.1172/JCI73416.) The epithelial-derived nasopharyngeal carcinoma (NPC) is a rare tumor in most of the world; however, it is common in southern China, northern Africa, and Alaska. NPC is often left undiagnosed and untreated until a late stage of disease. Furthermore, while radiation therapy is effective against this tumor, local recurrence due to radioresistance is an important clinical problem. In this issue, Liu et al. report on their identification of the IL-6 family cytokine leukemia inhibitory factor (LIF) as a serum predictor of local NPC recurrence following radiation therapy. The authors developed this initial finding to discover a role for the LIF/LIFR/mTORC1 signaling axis in NPC tumor cell growth as well as radioresistance.
J Clin Invest. 2013 Nov 25:1-3. doi: 10.1172/JCI73416.) The epithelial-derived nasopharyngeal carcinoma (NPC) is a rare tumor in most of the world; however, it is common in southern China, northern Africa, and Alaska. NPC is often left undiagnosed and untreated until a late stage of disease. Furthermore, while radiation therapy is effective against this tumor, local recurrence due to radioresistance is an important clinical problem. In this issue, Liu et al. report on their identification of the IL-6 family cytokine leukemia inhibitory factor (LIF) as a serum predictor of local NPC recurrence following radiation therapy. The authors developed this initial finding to discover a role for the LIF/LIFR/mTORC1 signaling axis in NPC tumor cell growth as well as radioresistance.
27. Valouev, A., Z. Weng, R. Sweeney, S. Varma, Q. T. Le, C. Kong, A. Sidow, and R. West. (2013) Discovery of recurrent structural variants in nasopharyngeal carcinoma. Genome Res (pdf 3889. must read. NPC Gene Fusion.) We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC), and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split-reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1 and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call “coupled inversion,” one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (3 cases), PTK2 (6 cases) and SP3 (2 cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues.
28. Wu, C. C., H. Y. Chuang, C. Y. Lin, Y. J. Chen, W. H. Tsai, C. Y. Fang, S. Y. Huang, F. Y. Chuang, S. F. Lin, Y. Chang, and J. Y. Chen. (2013) Inhibition of epstein-barr virus reactivation in nasopharyngeal carcinoma cells by dietary sulforaphane. Molecular carcinogenesis 52, 946-58. (pdf 3564.) Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcingenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotocixity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation. (c) 2012 Wiley Periodicals, Inc.
29. Xi, M., S. L. Liu, L. Zhao, J. X. Shen, L. Zhang, P. Zhang, and M. Z. Liu. (2013) Prognostic factors and survival in patients with radiation-related second malignant neoplasms following radiotherapy for nasopharyngeal carcinoma. PLoS One 8, e84586. (pdf not yet) PURPOSE: To analyze the clinicopathological characteristics, treatment modalities, and potential prognostic factors of radiation-related second malignant neoplasms (SMNs) in a large group of nasopharyngeal carcinoma (NPC) cases. METHODS AND MATERIALS: Institutional electronic medical records of 39,118 patients with NPC treated by definitive radiotherapy between February 1964 and December 2003 were reviewed. A total of 247 patients with confirmed SMN attributable to radiotherapy were included. RESULTS: Median latency between radiotherapy for NPC and the diagnosis of SMN was 9.5 years (range, 3.1-36.8 years). Squamous cell carcinoma was the most common histologic type, followed by fibrosarcoma and adenocarcinoma. Median progression-free survival and overall survival (OS) of the 235 patients who underwent treatment were 17.3 months and 28.5 months, respectively. The 5-year OS rates were 42.9%, 23.7%, and 0% for the surgery, radiotherapy, and chemotherapy groups, respectively. The independent prognostic factors associated with survival were sex, histologic type, and treatment modality in both the early stage subgroup and the advanced stage subgroup of SMN. CONCLUSIONS: Sex, histologic type, and treatment modality were the significant prognostic factors for SMN. Complete resection offers the best chance for long-term survival. In select patients with locally advanced and unresectable SMN, reirradiation should be strongly considered as a curative option.
30. Yip, Y. L., P. S. Pang, W. Deng, C. M. Tsang, M. Zeng, P. M. Hau, C. Man, Y. Jin, A. P. Yuen, and S. W. Tsao. (2013) Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study. PLoS One 8, e78395. (pdf 3819. Tuesday Journal Club by Ingrid.) Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.
31 Szeto, C. Y., C. H. Lin, S. C. Choi, T. T. Yip, R. K. Ngan, G. S. Tsao, and M. Li Lung. Integrated mRNA and microRNA transcriptome sequencing characterizes sequence variants and mRNA-microRNA regulatory network in nasopharyngeal carcinoma model systems. (2014). FEBS open bio. 4: 128-40. pdf 3964 Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia among the Chinese population. Aberrant regulation of transcripts has been implicated in many types of cancers including NPC. Herein, we characterized mRNA and miRNA transcriptomes by RNA sequencing (RNASeq) of NPC model systems. Matched total mRNA and small RNA of undifferentiated Epstein-Barr virus (EBV)-positive NPC xenograft X666 and its derived cell line C666, well-differentiated NPC cell line HK1, and the immortalized nasopharyngeal epithelial cell line NP460 were sequenced by Solexa technology. We found 2812 genes and 149 miRNAs (human and EBV) to be differentially expressed in NP460, HK1, C666 and X666 with RNASeq; 533 miRNA-mRNA target pairs were inversely regulated in the three NPC cell lines compared to NP460. Integrated mRNA/miRNA expression profiling and pathway analysis show extracellular matrix organization, Beta-1 integrin cell surface interactions, and the PI3K/AKT, EGFR, ErbB, and Wnt pathways were potentially deregulated in NPC. Real-time quantitative PCR was performed on selected mRNA/miRNAs in order to validate their expression. Transcript sequence variants such as short insertions and deletions (INDEL), single nucleotide variant (SNV), and isomiRs were characterized in the NPC model systems. A novel TP53 transcript variant was identified in NP460, HK1, and C666. Detection of three previously reported novel EBV-encoded BART miRNAs and their isomiRs were also observed. Meta-analysis of a model system to a clinical system aids the choice of different cell lines in NPC studies. This comprehensive characterization of mRNA and miRNA transcriptomes in NPC cell lines and the xenograft provides insights on miRNA regulation of mRNA and valuable resources on transcript variation and regulation in NPC, which are potentially useful for mechanistic and preclinical studies.
32. Kwok, H., C. W. Wu, A. L. Palser, P. Kellam, P. C. Sham, D. L. Kwong, and A. K. Chiang. (2014) Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsies. J Virol (pdf 4094. MS format.) Undifferentiated nasopharyngeal carcinoma (NPC) has 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date and the role of EBV genomic variations in the pathogenesis of NPC is unclear. Our project aimed to obtain the sequences of EBV genomes in multiple NPC biopsies in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first re-sequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization followed by next generation sequencing, de novo assembly and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy-derived EBV genomes, designated HKNPC2 to -9, were then determined. They harbored 1736 variations in total, including 1601 substitutions, 64 insertions and 71 deletions, in comparison with the reference EBV. Furthermore, genes encoding latent, early lytic, tegument and glyco-proteins were found to contain non-synonymous mutations of potential biological significance. Phylogenetic analysis showed that HKNPC6 and -7 genomes, which were isolated from tumor biopsies of advanced metastatic NPC cases, to be distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater- sequence diversity among EBV isolates derived from NPC biopsies is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis. IMPORTANCE: This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsies and revealed the sequence diversity on a whole genome level among these EBV isolates. At least two lineages of EBV strains are observed and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.
33. Lin, D. C., X. Meng, M. Hazawa, Y. Nagata, A. M. Varela, L. Xu, Y. Sato, L. Z. Liu, L. W. Ding, A. Sharma, B. C. Goh, S. C. Lee, B. F. Petersson, F. G. Yu, P. Macary, M. Z. Oo, C. S. Ha, H. Yang, S. Ogawa, K. S. Loh, and H. P. Koeffler. (2014) The genomic landscape of nasopharyngeal carcinoma. Nat Genet (pdf 4083. Must read.) Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC.
34. Lung, H. L., O. Y. Man, M. C. Yeung, J. M. Ko, A. K. Cheung, E. W. Law, Z. Yu, W. H. Shuen, E. Tung, S. H. Chan, D. K. Bangarusamy, Y. Cheng, X. Yang, R. Kan, Y. Phoon, K. C. Chan, D. Chua, D. L. Kwong, A. W. Lee, M. F. Ji, and M. L. Lung. (2014) SAA1 polymorphisms are associated with variation in antiangiogenic and tumor-suppressive activities in nasopharyngeal carcinoma. Oncogene (pdf 3990.) Nasopharyngeal carcinoma (NPC) is a cancer that occurs in high frequency in Southern China. A previous functional complementation approach and the subsequent cDNA microarray analysis have identified that serum amyloid A1 (SAA1) is an NPC candidate tumor suppressor gene. SAA1 belongs to a family of acute-phase proteins that are encoded by five polymorphic coding alleles. The SAA1 genotyping results showed that only three SAA1 isoforms (SAA1.1, 1.3 and 1.5) were observed in both Hong Kong NPC patients and healthy individuals. This study aims to determine the functional role of SAA1 polymorphisms in tumor progression and to investigate the relationship between SAA1 polymorphisms and NPC risk. Indeed, we have shown that restoration of SAA1.1 and 1.3 in the SAA1-deficient NPC cell lines could suppress tumor formation and angiogenesis in vitro and in vivo. The secreted SAA1.1 and SAA1.3 proteins can block cell adhesion and induce apoptosis in the vascular endothelial cells. In contrast, the SAA1.5 cannot induce apoptosis or inhibit angiogenesis because of its weaker binding affinity to alphaVbeta3 integrin. This can explain why SAA1.5 has no tumor-suppressive effects. Furthermore, the NPC tumors with this particular SAA1.5/1.5 genotype showed higher levels of SAA1 gene expression, and SAA1.1 and 1.3 alleles were preferentially inactivated in tumor tissues that were examined. These findings further strengthen the conclusion for the defective function of SAA1.5 in suppression of tumor formation and angiogenesis. Interestingly, the frequency of the SAA1.5/1.5 genotype in NPC patients was ~2-fold higher than in the healthy individuals (P=0.00128, odds ratio=2.28), which indicates that this SAA1 genotype is significantly associated with a higher NPC risk. Collectively, this homozygous SAA1.5/1.5 genotype appears to be a recessive susceptibility gene, which has lost the antiangiogenic function, whereas SAA1.1 and SAA1.3 are the dominant alleles of the tumor suppressor phenotype.Oncogene advance online publication, 10 March 2014; doi:10.1038/onc.2014.12.
35. Strong, M. J., M. Baddoo, A. Nanbo, M. Xu, A. Puetter, and Z. Lin. (2014) Comprehensive RNA-seq analysis reveals contamination of multiple nasopharyngeal carcinoma cell lines with HeLa cell genomes. J Virol (pdf 4091. MS format.) In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPC), we employed our RNA-seq analysis pipeline, RNA CoMPASS to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC/EBV researchers. Sequencing datasets from both CNE1 and HONE1 were found to contain reads for human papillomavirus (HPV-18). Subsequent real-time RT-PCR analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed identical patterns to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, and HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell specific L1 retrotransposon insertion, suggesting these three HPV-18+ NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq based gene level SNV analysis. Taken together, we conclude a widespread HeLa contamination may exist in many NPC cell lines and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq based approach for cell authentication. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of the Epstein-Barr virus (EBV). Using an RNA-seq based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.
36. Wen, Q., J. Li, W. Wang, G. Xie, L. Xu, J. Luo, S. Chu, L. She, D. Li, D. Huang, and S. Fan. (2014) Increased Expression of Flotillin-2 Protein as a Novel Biomarker for Lymph Node Metastasis in Nasopharyngeal Carcinoma. PLoS One 9, e101676. (pdf not yet) 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="" 93="" a="" analysis.="" be="" biomarker="" by="" can="" clinical="" correlated="" expression="" factor="" flot-2="" for="" higher="" in="" increased="" increasing="" independent="" is="" lymph="" metastases="" metastasis.="" metastasis="" multivariate="" node="" novel="" npc.="" npc="" obviously="" of="" our="" p="" patients.="" patients="" percentage="" positive="" predict="" predicted="" predicting="" protein="" proved="" respectively="" results="" sensitivity="" significantly="" stages="" suggest="" taken="" than="" that="" the="" those="" to="" together="" was="" with="" without="">
37. Yang, C. H., H. L. Wang, Y. S. Lin, K. P. Kumar, H. C. Lin, C. J. Chang, C. C. Lu, T. T. Huang, J. Martel, D. M. Ojcius, Y. S. Chang, J. D. Young, and H. C. Lai. (2014) Identification of CD24 as a Cancer Stem Cell Marker in Human Nasopharyngeal Carcinoma. PLoS One 9, e99412. (pdf not yet Yang, Chun-Hung, Wang, Hui-Ling, ..Young, John D, Lai, Hsin-Chih) Cancer stem cells (CSCs) represent a unique sub-population of tumor cells with the ability to initiate tumor growth and sustain self-renewal. Although CSC biomarkers have been described for various tumors, only a few markers have been identified for nasopharyngeal carcinoma (NPC). In this study, we show that CD24+ cells isolated from human NPC cell lines express stem cell genes (Sox2, Oct4, Nanog, Bmi-1, and Rex-1), and show activation of the Wnt/beta-catenin signaling pathway. CD24+ cells possess typical CSC characteristics that include enhanced cell proliferation, increased colony and sphere formation, maintenance of cell differentiation potential in prolonged culture, and enhanced resistance to chemotherapeutic drugs. Notably, CD24+ cells produce tumors following inoculation of as few as 500 cells in immunodeficient NOD/SCID mice. CD24+ cells further show increased invasion ability in vitro, which correlates with enhanced expression of matrix metalloproteinase 2 and 9. In summary, our results suggest that CD24 represents a novel CSC biomarker in NPC.
38. Ye, S. B., Z. L. Li, D. H. Luo, B. J. Huang, Y. S. Chen, X. S. Zhang, J. Cui, Y. X. Zeng, and J. Li. (2014) Tumor-derived exosomes promote tumor progression and T-cell dysfunction through the regulation of enriched exosomal microRNAs in human nasopharyngeal carcinoma. Oncotarget (Ye, Shu-Biao Li, Ze-Lei ,Zeng, Yi-Xin, Li, Jiang) Tumor-derived exosomes contain biologically active proteins and messenger and microRNAs (miRNAs). These particles serve as vehicles of intercellular communication and are emerging mediators of tumorigenesis and immune escape. Here, we isolated 30-100 nm exosomes from the serum of patients with nasopharyngeal carcinoma (NPC) or the supernatant of TW03 cells. Increased circulating exosome concentrations were correlated with advanced lymphoid node stage and poor prognosis in NPC patients (P< 0.05). TW03-derived exosomes impaired T-cell function by inhibiting T-cell proliferation and Th1 and Th17 differentiation and promoting Treg induction by NPC cells in vitro. These results are associated with decreases in ERK, STAT1, and STAT3 phosphorylation and increases in STAT5 phosphorylation in exosome-stimulated T-cells. TW03-derived exosomes increased the proinflammatory cytokines IL-1beta, IL-6, and IL-10 but decreased IFNgamma, IL-2, and IL-17 release from CD4+ or CD8+ T-cells. Furthermore, five commonly over-expressed miRNAs were identified in the exosomes from patient sera or NPC cells: hsa-miR-24-3p, hsa-miR-891a, hsa-miR-106a-5p, hsa-miR-20a-5p, and hsa-miR-1908. These over-expressed miRNA clusters down-regulated the MARK1 signaling pathway to alter cell proliferation and differentiation. Overall, these observations reveal the clinical relevance and prognostic value of tumor-derived exosomes and identify a unique intercellular mechanism mediated by tumor-derived exosomes to modulate T-cell function in NPC.