RESUMO
Epstein-Barr virus (EBV) induces histone modifications to regulate signaling pathways involved in EBV-driven tumorigenesis. To date, the regulatory mechanisms involved are poorly understood. In this study, we show that EBV infection of epithelial cells is associated with aberrant histone modification; specifically, aberrant histone bivalent switches by reducing the transcriptional activation histone mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) at the promoter regions of a panel of DNA damage repair members in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA damage repair family members in base excision repair (BER), homologous recombination, nonhomologous end-joining, and mismatch repair (MMR) pathways showed aberrant histone bivalent switches. Among this panel of DNA repair members, MLH1, involved in MMR, was significantly down-regulated in EBV-infected NPE cells through aberrant histone bivalent switches in a promoter hypermethylation-independent manner. Functionally, expression of MLH1 correlated closely with cisplatin sensitivity both in vitro and in vivo. Moreover, seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched in pathway analysis in a promoter hypermethylation-independent manner. This observation is further validated by their down-regulation in EBV-infected NPE cells. The in vitro comet and apurinic/apyrimidinic site assays further confirmed that EBV-infected NPE cells showed reduced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner.
Assuntos
Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Código das Histonas/genética , Histonas/genética , Ilhas de CpG/genética , Dano ao DNA/genética , Metilação de DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Regulação da Expressão Gênica/genética , Herpesvirus Humano 4/patogenicidade , Recombinação Homóloga/genética , Humanos , Proteína 1 Homóloga a MutL/genética , Nasofaringe/crescimento & desenvolvimento , Nasofaringe/patologia , Nasofaringe/virologia , Regiões Promotoras GenéticasRESUMO
Multiple factors, including host genetics, environmental factors, and Epstein-Barr virus (EBV) infection, contribute to nasopharyngeal carcinoma (NPC) development. To identify genetic susceptibility genes for NPC, a whole-exome sequencing (WES) study was performed in 161 NPC cases and 895 controls of Southern Chinese descent. The gene-based burden test discovered an association between macrophage-stimulating 1 receptor (MST1R) and NPC. We identified 13 independent cases carrying the MST1R pathogenic heterozygous germ-line variants, and 53.8% of these cases were diagnosed with NPC aged at or even younger than 20 y, indicating that MST1R germline variants are relevant to disease early-age onset (EAO) (age of ≤20 y). In total, five MST1R missense variants were found in EAO cases but were rare in controls (EAO vs. control, 17.9% vs. 1.2%, P = 7.94 × 10(-12)). The validation study, including 2,160 cases and 2,433 controls, showed that the MST1R variant c.G917A:p.R306H is highly associated with NPC (odds ratio of 9.0). MST1R is predominantly expressed in the tissue-resident macrophages and is critical for innate immunity that protects organs from tissue damage and inflammation. Importantly, MST1R expression is detected in the ciliated epithelial cells in normal nasopharyngeal mucosa and plays a role in the cilia motility important for host defense. Although no somatic mutation of MST1R was identified in the sporadic NPC tumors, copy number alterations and promoter hypermethylation at MST1R were often observed. Our findings provide new insights into the pathogenesis of NPC by highlighting the involvement of the MST1R-mediated signaling pathways.
Assuntos
Exoma , Predisposição Genética para Doença , Neoplasias Nasofaríngeas/genética , Receptores Proteína Tirosina Quinases/genética , Análise de Sequência , Adolescente , Adulto , Carcinoma , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Adulto JovemRESUMO
Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with a unique geographical distribution. The genomic abnormalities leading to NPC pathogenesis remain unclear. In total, 135 NPC tumors were examined to characterize the mutational landscape using whole-exome sequencing and targeted resequencing. An APOBEC cytidine deaminase mutagenesis signature was revealed in the somatic mutations. Noticeably, multiple loss-of-function mutations were identified in several NF-κB signaling negative regulators NFKBIA, CYLD, and TNFAIP3 Functional studies confirmed that inhibition of NFKBIA had a significant impact on NF-κB activity and NPC cell growth. The identified loss-of-function mutations in NFKBIA leading to protein truncation contributed to the altered NF-κB activity, which is critical for NPC tumorigenesis. In addition, somatic mutations were found in several cancer-relevant pathways, including cell cycle-phase transition, cell death, EBV infection, and viral carcinogenesis. These data provide an enhanced road map for understanding the molecular basis underlying NPC.
Assuntos
Carcinoma/genética , Sequenciamento do Exoma/métodos , Mutação com Perda de Função/genética , NF-kappa B/metabolismo , Neoplasias Nasofaríngeas/genética , Transdução de Sinais/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Taxa de Mutação , Inibidor de NF-kappaB alfa/metabolismo , Carcinoma NasofaríngeoRESUMO
Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at â¼0.5 to 5 nM (versus â¼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21(WAF1) , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21(WAF1) and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers.
Assuntos
Antivirais/farmacologia , Depsipeptídeos/farmacologia , Ganciclovir/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Replicação Viral/efeitos dos fármacos , Acetilação , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinoma , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Histona Desacetilases/genética , Histonas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Proteína Quinase C-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia , Ativação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor suppressor genes (TSGs) play a prominent role in cancer and are important in the development of nasopharyngeal carcinoma (NPC), which is endemic in Southern China as well as Southeast Asia. Apart from TSGs, aberrant signalling pathways are also commonly associated with tumor progression. Unsurprisingly, the NF-κB pathway is frequently associated with angiogenesis and promoting tumor growth and development. Functional complementation studies using microcell-mediated chromosome transfer helped to identify IKBB as a putative TSG in NPC. IKBB, an inhibitor of NF-κB, has recently been shown to be inversely associated with tumor growth and metastasis via inactivation of the NF-κB pathway, but its suppressive role is still only poorly understood. This study takes the lead in revealing the suppressive role of IKBB in NPC. IKBB is silenced in the majority of NPC tumor tissues in all stages. Its suppressive role is substantiated by perturbation in tumor formation, cell migration and angiogenesis. Interestingly, IKBB not only affects the 'seed', but also influences the 'soil' by downregulating the transcriptional level of proangiogenic factors Rantes, Upar, IL6, and IL8. For the first time, our data establish the importance of a novel tumor suppressive IKBB gene in abrogating angiogenesis in NPC via the NF-κB signalling pathway, which is likely mediated by crosstalk with the Akt/Gsk3ß signalling pathway.
Assuntos
Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Carcinoma , Linhagem Celular Tumoral , Movimento Celular/genética , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas I-kappa B/genética , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/mortalidade , Neoplasias Nasofaríngeas/patologia , Estadiamento de Neoplasias , Neovascularização Patológica/genética , Prognóstico , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Chromosome 14 was transferred into tumorigenic nasopharyngeal carcinoma and esophageal carcinoma cell lines by a microcell-mediated chromosome transfer approach. Functional complementation of defects present in the cancer cells suppressed tumor formation. A candidate tumor-suppressor gene, cysteine-rich intestinal protein 2 (CRIP2), located in the hot spot for chromosomal loss at 14q32.3, was identified as an important candidate gene capable of functionally suppressing tumor formation. Previous studies have shown that CRIP2 is associated with development. To date, no report has provided functional evidence supporting a role for CRIP2 in tumor development. The present study provides unequivocal evidence that CRIP2 can functionally suppress tumorigenesis. CRIP2 is significantly down-regulated in nasopharyngeal carcinoma cell lines and tumors. CRIP2 reexpression functionally suppresses in vivo tumorigenesis and angiogenesis; these effects are induced by its transcription-repressor capability. It interacts with the NF-κB/p65 to inhibit its DNA-binding ability to the promoter regions of the major proangiogenesis cytokines critical for tumor progression, including IL6, IL8, and VEGF. In conclusion, we provide compelling evidence that CRIP2 acts as a transcription repressor of the NF-κB-mediated proangiogenic cytokine expression and thus functionally inhibits tumor formation and angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transformação Celular Neoplásica/genética , Citocinas/genética , NF-kappa B/metabolismo , Neovascularização Patológica/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Proteínas Angiogênicas/análise , Linhagem Celular , Linhagem Celular Tumoral , Cromossomos Humanos Par 14 , Citocinas/fisiologia , Humanos , Proteínas com Domínio LIM , Proteínas Repressoras/fisiologiaRESUMO
The interplay between host cell genetics and Epstein-Barr virus (EBV) infection contributes to the development of nasopharyngeal carcinoma (NPC). Understanding the host genetic and epigenetic alterations and the influence of EBV on cell signaling and host gene regulation will aid in understanding the molecular pathogenesis of NPC and provide useful biomarkers and targets for diagnosis and therapy. In this review, we provide an update of the oncogenes and tumor suppressor genes associated with NPC, as well as genes associated with NPC risk including those involved in carcinogen detoxification and DNA repair. We also describe the importance of host genetics that govern the human leukocyte antigen (HLA) complex and immune responses, and we describe the impact of EBV infection on host cell signaling changes and epigenetic regulation of gene expression. High-power genomic sequencing approaches are needed to elucidate the genetic basis for inherited susceptibility to NPC and to identify the genes and pathways driving its molecular pathogenesis.
Assuntos
Epigênese Genética , Infecções por Vírus Epstein-Barr , Predisposição Genética para Doença , Neoplasias Nasofaríngeas/etiologia , Carcinoma , Genes Supressores de Tumor , Herpesvirus Humano 4/genética , Humanos , Carcinoma Nasofaríngeo , Oncogenes , Transdução de SinaisRESUMO
The identification of cancer genes in sporadic cancers has been recognized as a major challenge in the field. It is clear that deletion mapping, genomic sequencing, comparative genomic hybridization, or global gene expression profiling alone would not have easily identified candidate tumor suppressor genes (TSGs) from the huge array of lost regions or genes observed in nasopharyngeal carcinoma (NPC). In addition, the epigenetically silenced genes would not have been recognized by the mapping of deleted regions. In this review, we describe how functional approaches using monochromosome transfer may be used to circumvent the above problems and identify TSGs in NPC. A few examples of selected NPC TSGs and their functional roles are reviewed. They regulate a variety of gene functions including cell growth and proliferation, adhesion, migration, invasion, epithelial-mesenchymal transition, metastasis, and angiogenesis. These studies show the advantages of using functional approaches for identification of TSGs.
Assuntos
Mapeamento Cromossômico/métodos , Predisposição Genética para Doença/genética , Neoplasias Nasofaríngeas/genética , Proteínas Supressoras de Tumor/genética , Animais , Carcinoma , Fusão Celular/métodos , Movimento Celular/genética , Humanos , Células Híbridas/metabolismo , Camundongos , Carcinoma NasofaríngeoRESUMO
BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/ß-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological ß-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of ß-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells. RESULTS: Introduction of increased ß-catenin signaling, haploid expression of ß-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/ß-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous ß-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/ß-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-ß, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using ß-catenin shRNA inhibitory assays, a dominant role for ß-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres. CONCLUSIONS: Wnt/ß-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/ß-catenin signaling with stemness transition networks.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Nasofaríngeas/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/genética , beta Catenina/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Carcinoma , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células Híbridas/metabolismo , Células Híbridas/patologia , Fator 4 Semelhante a Kruppel , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
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.
Assuntos
Cromossomos Humanos Par 14/genética , Neoplasias Nasofaríngeas/genética , Proteínas Supressoras de Tumor/genética , Animais , Ciclo Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Metilação de DNA , Citometria de Fluxo , Imunofluorescência , Técnicas de Transferência de Genes , Genes Supressores de Tumor , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Nus , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Reação em Cadeia da Polimerase/métodos , Transfecção/instrumentação , Transfecção/métodos , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismoRESUMO
THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)/G(1) phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC.
Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Neoplasias Nasofaríngeas/patologia , Antígenos Thy-1/fisiologia , Animais , Western Blotting , Adesão Celular , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
By using a functional complementation approach, suppression of tumorigenicity was observed after transfer of intact or truncated copies of chromosome 3 into a nasopharyngeal carcinoma (NPC) HONE1 cell line. The extra exogenous chromosome 3 in the microcell hybrids (MCHs) significantly extended the lag period of tumor formation, which may be associated with loss or inactivation of wild type alleles from the normal donor chromosome 3. Representative tumors, which grew in nude mice were reconstituted into culture and expanded as tumor segregants (TSs). In our study, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9), a gene mapping to 3p14.2, was identified to be critically associated with tumor suppression in NPC. Gene expression analysis showed that ADAMTS9 was either not expressed or was downregulated in HONE1 cells, TSs and NPC cell lines. The mechanism of ADAMTS9 gene inactivation in the NPC cell lines and tissues was attributed to promoter hypermethylation. Using a tissue microarray and immunohistochemical staining, 31 of 66 (47%) of the NPC cases showed downregulated or absence of ADAMTS9 expression. ADAMTS9 expression was downregulated or lost in 17 of 23 (73.9%) lymph node metastatic NPC specimens, which was significantly higher than in 14 of 43 (32.6%) primary tumors. After transfection of the ADAMTS9 gene into 7 NPC cell lines, a dramatic reduction of colony forming ability was observed. These findings support ADAMTS9 as a putative tumor suppressor gene in vivo in NPC that is significantly associated with lymph node metastases.
Assuntos
Proteínas ADAM/genética , Carcinoma/genética , Linfonodos/patologia , Neoplasias Nasofaríngeas/genética , Proteína ADAMTS9 , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma/secundário , Cromossomos Humanos Par 3 , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genótipo , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Análise em Microsséries , Repetições de Microssatélites , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In previous studies, we successfully refined nasopharyngeal carcinoma (NPC) critical regions (CRs) mapping to chromosome 11q13 and 11q22-23. The chromosome 11 fragment containing the 1.8 Mb NPC CR at 11q13 (CR1), the CR at 11q22.3 mapped near D11S2000 (CR2), part of the CR at 11q23.1-11q23.2 overlapping with D11S1300 and D11S1391 (CR3), and the CR at cell adhesion molecule 1 (CADM1) locus (CR4), was chosen as the chromosome 11 donor cell line for the present study. Gamma irradiation was applied to cleave this truncated chromosome into smaller fragments and a new panel of donor cells containing further deleted fragments was produced. Subclones XMCH3.2 and XMCH3.4 were chosen for subsequent transfer to HONE1 cells; each contains a single copy of deleted chromosome 11 fragment with or without CR2 and the THY1 locus, previously shown to be involved in NPC. Both resultant chromosome 11 fragments in XMCH3.2 and XMCH3.4 caused tumor suppression. The association of alpha B-crystallin (CRYAB), a gene identified as being differentially expressed by gene profiling of NPC and an immortalized nasopharyngeal epithelial cell line, and which is located near CR3, was found to be associated with tumor suppression in all the tumor-suppressive hybrids. In addition, the expression level of this gene was down-regulated in the 7 NPC cell lines and in 5 out of 14 normal/tumor tissue pairs in the present study. Both promoter hypermethylation and allelic loss may be involved in the inactivation of this gene, suggesting its possible role in NPC development.
Assuntos
Cromossomos Humanos Par 11 , Neoplasias Nasofaríngeas/genética , Cadeia B de alfa-Cristalina/fisiologia , Animais , Sequência de Bases , Metilação de DNA , Primers do DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Repetições de Microssatélites/genética , Neoplasias Nasofaríngeas/patologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Nidogen-2 (NID2), a secretory basement membrane protein, has been implicated as a potential biomarker in ovarian cancer and hepatocellular carcinoma. OBJECTIVE: In this study, we aimed to investigate the utility of detecting serum NID2 levels for identification of esophageal squamous cell carcinoma (ESCC) patients and prediction of poor survival outcome. METHODS: Using an in-house NID2 enzyme-linked immunosorbent assay (ELISA), serum samples from 101 ESCC patients and 50 healthy controls were screened for their NID2 levels. RESULTS: The serum NID2 levels in ESCC patients (median 24.4 µg/L) are significantly higher (p= 4.3e-09) than that of the healthy controls (median 15.85 µg/L). The receiver operating characteristic (ROC) curve demonstrated an area under the curve of 0.756. At the threshold of 17.95 µg/L, the sensitivity and specificity achieved are 0.76 and 0.63, respectively. Kaplan-Meier survival analysis revealed that patients with high serum NID2 levels (⩾ 32.6 µg/L) have significantly higher risk of death (HR = 1.984, 95% CI: 1.175-3.349; log-rank p-value = 0.012) compared to those with low serum NID2 levels (< 20.0 µg/L). CONCLUSIONS: In conclusion, we show that detecting the elevation of serum NID2 levels has potential diagnostic and prognostic value for ESCC patients.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/mortalidade , Moléculas de Adesão Celular/sangue , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/mortalidade , Adulto , Idoso , Proteínas de Ligação ao Cálcio , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Carcinoma de Células Escamosas do Esôfago , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Curva ROCRESUMO
Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22-23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.
Assuntos
Carcinoma/genética , Neoplasias Nasofaríngeas/genética , Antígenos Thy-1/genética , Carcinoma/patologia , Cromossomos Humanos Par 11 , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Humanos , Linfonodos/patologia , Metástase Linfática/genética , Análise em Microsséries , Neoplasias Nasofaríngeas/patologia , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Thy-1/metabolismo , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy that shows a remarkable geographical and ethic distribution. Multiple factors including predisposing genetic factors, environmental carcinogens, and Epstein-Barr virus (EBV) infection contribute to the accumulation of genetic and epigenetic alterations leading to NPC development. Emerging technologies now allow us to detailedly characterize and understand cancer genomes. Genome-wide studies show that typically NPC tumors are characterized as having comparatively low mutation rates, widespread hypermethylation, and frequent copy number alterations and chromosome abnormalities. In this review, we provide an updated overview of the genetic and epigenetic aberrations that likely drive nasopharyngeal tumor development and progression. We integrate the previous knowledge and novel findings from whole-exome sequencing (WES) and methylome studies in NPC, and further discuss the potential use of these findings to identify biomarkers for NPC diagnosis and prognosis.
Assuntos
Epigênese Genética , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/genética , Biomarcadores Tumorais/genética , Carcinoma , Metilação de DNA , Infecções por Vírus Epstein-Barr/genética , Dosagem de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Perda de Heterozigosidade , Mutação , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/virologia , PrognósticoRESUMO
Nidogen-2 (NID2) is a key component of the basement membrane that stabilizes the extracellular matrix (ECM) network. The aim of the study is to analyze the functional roles of NID2 in the pathogenesis of nasopharyngeal carcinoma (NPC) and esophageal squamous cell carcinoma (ESCC). We performed genome-wide methylation profiling of NPC and ESCC and validated our findings using the methylation-sensitive high-resolution melting (MS-HRM) assay. Results showed that promoter methylation of NID2 was significantly higher in NPC and ESCC samples than in their adjacent non-cancer counterparts. Consistently, down-regulation of NID2 was observed in the clinical samples and cell lines of both NPC and ESCC. Re-expression of NID2 suppresses clonogenic survival and migration abilities of transduced NPC and ESCC cells. We showed that NID2 significantly inhibits liver metastasis. Mechanistic studies of signaling pathways also confirm that NID2 suppresses the EGFR/Akt and integrin/FAK/PLCγ metastasis-related pathways. This study provides novel insights into the crucial tumor metastasis suppression roles of NID2 in cancers.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma/genética , Moléculas de Adesão Celular/genética , Metilação de DNA , Neoplasias Esofágicas/genética , Inativação Gênica , Neoplasias Nasofaríngeas/genética , Animais , Proteínas de Ligação ao Cálcio , Carcinoma/metabolismo , Carcinoma/secundário , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundário , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Receptores ErbB/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Integrinas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica , Fosfolipase C gama/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga TumoralRESUMO
Protein Tyrosine Phosphatase, Receptor Type G (PTPRG) was identified as a candidate tumor suppressor gene in nasopharyngeal carcinoma (NPC). PTPRG induces significant in vivo tumor suppression in NPC. We identified EGFR as a PTPRG potential interacting partner and examined this interaction. Dephosphorylation of EGFR at EGFR-Y1068 and -Y1086 sites inactivated the PI3K/Akt signaling cascade and subsequent down-regulation of downstream pro-angiogenic and -invasive proteins (VEGF, IL6, and IL8) and suppressed tumor cell proliferation, angiogenesis, and invasion. The effect of Akt inhibition in NPC cells was further validated by Akt knockdown experiments in the PTPRG-down-regulated NPC cell lines. Our results suggested that inhibition of Akt in NPC cells induces tumor suppression at both the in vitro and in vivo levels, and also importantly, in vivo metastasis. In conclusion, we confirmed the vital role of PTPRG in inhibiting Akt signaling with the resultant suppression of in vivo tumorigenesis and metastasis.
Assuntos
Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Animais , Carcinogênese , Carcinoma , Linhagem Celular Tumoral , Proliferação de Células , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Transdução de SinaisRESUMO
NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC). Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM) protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-ß Binding Protein 2 (LTBP2) protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development.
Assuntos
Carcinoma/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , NF-kappa B/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Fator de Transcrição RelA/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Nasofaríngeas/patologia , Fosforilação , Transdução de Sinais , Microambiente TumoralRESUMO
Altered patterns of DNA methylation are key features of cancer. Nasopharyngeal carcinoma (NPC) has the highest incidence in Southern China. Aberrant methylation at the promoter region of tumor suppressors is frequently reported in NPC; however, genome-wide methylation changes have not been comprehensively investigated. Therefore, we systematically analyzed methylome data in 25 primary NPC tumors and nontumor counterparts using a high-throughput approach with the Illumina HumanMethylation450 BeadChip. Comparatively, we examined the methylome data of 11 types of solid tumors collected by The Cancer Genome Atlas (TCGA). In NPC, the hypermethylation pattern was more dominant than hypomethylation and the majority of de novo methylated loci were within or close to CpG islands in tumors. The comparative methylome analysis reveals hypermethylation at chromosome 6p21.3 frequently occurred in NPC (false discovery rate; FDR=1.33 × 10(-9) ), but was less obvious in other types of solid tumors except for prostate and Epstein-Barr virus (EBV)-positive gastric cancer (FDR<10(-3) ). Bisulfite pyrosequencing results further confirmed the aberrant methylation at 6p in an additional patient cohort. Evident enrichment of the repressive mark H3K27me3 and active mark H3K4me3 derived from human embryonic stem cells were found at these regions, indicating both DNA methylation and histone modification function together, leading to epigenetic deregulation in NPC. Our study highlights the importance of epigenetic deregulation in NPC. Polycomb Complex 2 (PRC2), responsible for H3K27 trimethylation, is a promising therapeutic target. A key genomic region on 6p with aberrant methylation was identified. This region contains several important genes having potential use as biomarkers for NPC detection.