RESUMEN

Graft-versus-host disease (GVHD), a life-threatening complication after bone marrow transplantation (BMT), is induced by activation of alloreactive donor T cells. Our previous study demonstrated that transplantation of myeloid differentiation factor 88 (MyD88)-deficient knockout (KO) bone marrow (BM) resulted in aggravation of GVHD. Here, to understand the cellular mechanism, we performed longitudinal in vivo imaging and flow cytometric analyses followed by transcriptome and functional examination of donor MyD88-KO BM progenies in GVHD hosts, using a major histocompatibility complex-matched but minor histocompatibility antigen-mismatched C57BL/6→BALB.B model. In GVHD hosts with MyD88-KO BMT, donor BM-derived CD11b+Gr-1+ cells were found to undergo cell death, a fate significantly different from the explosive expansion shown by the wild type (WT) counterparts, and also from the moderate expansion of the WT or MyD88-KO BM-derived cells in non-GVHD hosts. It was also revealed that MyD88-KO CD11b+Gr-1+ cells preferred differentiation into CD11c+ dendritic cells (DCs) to expansion as myeloid-derived suppressor cells in GVHD hosts or in high inflammatory in vitro conditions. These CD11c+ DCs comprised the majority of MyD88-KO CD11b+Gr-1+ apoptotic cells in GVHD hosts. Their ability to cross-present alloantigens of host origin contributed to the enhancement of T cell alloreactivity, causing GVHD aggravation and eventually death through the killing function of activated T cells. These results provide insights into the roles of MyD88 in myelopoiesis of donor BM and the protective effects in GVHD hosts, helpful information for development of a strategy to control GVHD.

RESUMEN

Tumor infiltrating lymphocytes (TIL) in Epstein-Barr virus (EBV)-associated/microsatellite-unstable (MSI) gastric carcinomas (GC) constitute immune-active principal cellular components of tumor microenvironment and contribute to better prognosis. With the remarkable success of cancer immunotherapies, there is an urgent need for a comprehensive understanding of tumor-immune interactions in patients with GC in the context of host immune response. To identify GC subtype-specific immune response gene set, we tested differentially expressed genes for MSI and EBV+ GC subtypes in randomly selected test set (n = 278) in merged ACRG-SMC microarray and TCGA RNA sequencing data set. We identified Host ImmunE Response index (HIERÏ) consisting of 29 immune genes classifying GC patients into robust 3 groups with prognostic significance. Immune-high cluster 1 was enriched with PD-L1High/EBV+/MSI/TILHigh with the best clinical outcome while immune-low cluster 3 displayed worst outcome and exemplified with PD-L1Low/EBV-/MSS. The results were validated in the same cohort (n = 279) and independent cohort (n = 181) with RNA from formalin-fixed paraffin-embedded (FFPE) tissue. Unexpectedly, nearly half of GC in cluster 1 were EBV-/MSS and 10% of cluster 3 GC were EBV+/MSI GC patients, suggesting that in addition to EBV+/MSI GC subtypes, EBV-/MSS subtype also constitutes almost half of high immune cluster and would be a good candidate for immune checkpoint inhibitor therapy. In contrary, almost 10% of EBV+/MSI GC patients may not respond to immune checkpoint inhibitor therapy. Thus, our HIERÏ gene signature demonstrates the potential to subclassify tumor immunity levels, predict prognosis and help immunotherapeutic decisions.

RESUMEN

Graft-versus-host disease (GHVD) is a severe complication after allogeneic hematopoietic stem cell transplantation. The degree of inflammation in the gastrointestinal tract, a major GVHD target organ, correlates with the disease severity. Intestinal inflammation is initiated by epithelial damage caused by pre-conditioning irradiation. In combination with damages caused by donor-derived T cells, such damage disrupts the epithelial barrier and exposes innate immune cells to pathogenic and commensal intestinal bacteria, which release ligands for Toll-like receptors (TLRs). Dysbiosis of intestinal microbiota and signaling through the TLR/myeloid differentiation primary response gene 88 (MyD88) pathways contribute to the development of intestinal GVHD. Understanding the changes in the microbial flora and the roles of TLR signaling in intestinal GVHD will facilitate the development of preventative and therapeutic strategies.

RESUMEN

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is one of the four subtypes of gastric carcinoma (GC), as defined by the novel classification recently proposed by The Cancer Genome Atlas. EBVaGC has several clinicopathological features such as longer survival and higher frequency of lymphoepithelioma-like carcinoma (LELC) and carcinoma with Crohn's disease-like lymphoid reaction that distinguish it from EBV-negative GC. The intensity and pattern of host cellular immune response in GC have been found to significantly correlate with the prognosis of patients with GC, suggesting that immune reaction and tumor microenvironment have critical roles in the progression of GC, and in particular, EBVaGC. Here, we reviewed the cellular and molecular mechanisms underlying prominent immune reactions in patients with EBVaGC. In EBVaGC, deregulation of the expression of immune response-related genes promotes marked intra- or peritumoral immune cell infiltration. The expression of programmed death receptor-ligand 1 is known to be increased in EBVaGC, and therefore, it has been proposed as a favorable prognostic factor for patients with EBVaGC, albeit some data supporting this claim are controversial. Overall, the underlying mechanisms and clinical significance of the host cellular immune response in patients with EBVaGC have not been thoroughly elucidated. Therefore, further research is necessary to better understand the role of tumor microenvironment in EBVaGC.

RESUMEN

Despite their suggested importance, the mechanistic roles of FGFR2 and gastric cancer stem cell (GCSC) marker CD44 remain unclear. We investigated cross talk between CD44 and FGFR2. FGFR2 and CD44 positively regulate each other's expression. While FGFR2 suppresses c-Myc transcription, CD44 activates it. c-Myc in turn augments FGFR2 transcription. CD44 knockdown (KD) depleted FGFR2 and other GCSC markers, decreased c-Myc and Sox2 expression, and suppressed tumor growth, whereas CD44 activation led to FGFR2 induction. FGFR2 KD decreased most GCSC marker expression, including CD44, but increased c-Myc and Sox2 expression and attenuated tumor growth. FGFR2 kinase inhibitor and FGFR2 neutralizing antibody decreased the CD44+/hi GCSC fraction. Conversely, FGFR2 overexpression increased CD44 and accelerated tumor growth in mice. FGFR2 was co-expressed and colocalized diffusively with CD44, EpCAM, and LGR5. In contrast, phospho-FGFR2 colocalized densely with CD44, forming an aggregated signaling complex that was prevented by FGFR2 inhibition. The c-Myc KD depleted FGFR2 but not CD44. Similarly to CD44+/hi phenotypes, sorted FGFR+/hi cells had larger volumes, formed more tumor spheres, grew faster in vivo with bigger tumor mass, and expressed more CD44, EpCAM, and HER2. These findings suggest that FGFR2+/hi cells have stemness properties. Moreover, in situ FGFR2 expression in patient-derived gastric cancer tissue correlated with tumorigenic potential in a xenograft model. In conclusion, CD44 and FGFR2 maintain stemness in gastric cancer by differentially regulating c-Myc transcription.

Asunto(s)

Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Hialuranos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias Gástricas/genética , Animales , Línea Celular Tumoral , Humanos , Receptores de Hialuranos/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Trasplante Heterólogo

RESUMEN

Functional inhibition of Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) can cause the death of EBV infected cells. In this study, a bioinformatics tool predicted the existence of putative extracellular signal-regulated kinase (ERK) docking and substrate consensus sites on EBNA1, suggesting that ERK2 could bind to and phosphorylate EBNA1. In accordance, ERK2 was found to phosphorylate EBNA1 serine 383 in a reaction suppressed by H20 (a structural congener of the ERK inhibitor), U0126 (an inhibitor of MEK kinase), and mutations at substrate (S383A) or putative ERK docking sites. Wild-type (S383) and phosphomimetic (S383D) EBNA1 demonstrated comparable transactivation function, which was suppressed by H20 or U0126. In contrast, non-phosphorylated EBNA1 mutants displayed significantly impaired transactivation activity. ERK2 knock-down by siRNA, or treatment with U0126 or H20 repressed EBNA1-dependent transactivation.Collectively, these data indicate that blocking ERK2-directed phosphorylation can suppress EBNA1-transactivation function in latent EBV-infected cells, validating ERK2 as a drug target for EBV-associated disorders.

Asunto(s)

Infecciones por Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Activación Transcripcional/fisiología , Latencia del Virus/fisiología , Línea Celular Tumoral , Herpesvirus Humano 4 , Humanos , Fosforilación , Serina/metabolismo

RESUMEN

Epstein Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) plays a pivotal in an EBV episome replication and persistence. Despite considerable attempts, there are no EBV drugs or vaccines. We attempted to eradicate EBV episomes by targeting EBNA1 using the transcription activator-like effector nucleases (TALEN) (E1TN). E1TN-mediated transient knockout (KO) of EBNA1 reduced EBNA1 expression, and caused significant loss of EBV genomes and progressive death of EBV-infected cells. Furthermore, when a mixture of EBV-infected Burkitt's lymphoma (BL) cells and EBV-negative BL cells was targeted by E1TN, EBV-negative cells were counter-selected while most EBV-infected cells died, further substantiating that EBNA1 KO caused selective death of EBV-infected cells. TALEN-mediated transient targeting of EBNA1 attenuated the growth of EBV-infected cells, implicating a possible therapeutic application of E1TN for EBV-associated disorders. [BMB Reports 2016; 49(4): 226-231].

Asunto(s)

Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Marcación de Gen , Herpesvirus Humano 4/fisiología , Muerte Celular , Línea Celular , Proliferación Celular , Dosificación de Gen , Técnicas de Inactivación de Genes , Genoma Viral , Herpesvirus Humano 4/genética , Humanos , Plásmidos , Transfección

RESUMEN

The prognostic effects of tumor infiltrating lymphocytes (TILs), especially regulatory T cells (Tregs) and myeloid derived suppressing cells (MDSCs) are inconclusive in gastric cancers. We investigated the frequencies of TILs including CD8+ T cells, CD45+CD4+CD25± FOXP3+ Tregs, CD45+CD11b+ CD14+ HLA-DR- MDSCs in 28 gastric cancer tissues by using multicolor flow cytometry. In gastric cancer tissue, the percentage of Tregs among the CD4+ T cell subset was substantially increased compared to that of Tregs among peripheral blood CD4+ T cells from the controls. High frequency of CD8+ T cells among CD3+ T cells correlated with increased overall survival (OS) (p = 0.005). High frequency of Tregs among CD4+ T cells correlated with increased OS (p < 0.001), and disease-free survival (DFS) (p = 0.039) and was an independent prognostic factor in OS (Hazard ratio: 0.047; 95% confidence interval, 0.006-0.372; p = 0.004). High frequency of MDSCs among total examined cells correlated with decreased OS (p = 0.027) and was an independent prognostic factor in OS (Hazard ratio 8.601; 95% confidence interval, 1.240-59.678; p = 0.029). We have demonstrated that high levels of Tregs among tumor-infiltrating CD4+ T cells were favorable, but an increased proportion of MDSCs was an adverse independent prognostic factor in gastric cancer. Our results may provide important insights for future immunotherapy in gastric cancer.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citometría de Flujo/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Células Supresoras de Origen Mieloide/inmunología , Recurrencia Local de Neoplasia/inmunología , Neoplasias Gástricas/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Estudios de Casos y Controles , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/patología , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Estadificación de Neoplasias , Pronóstico , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Tasa de Supervivencia , Linfocitos T Reguladores/patología , Células Tumorales Cultivadas

RESUMEN

Epstein-Barr virus (EBV) infection causes both Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34(+) hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted (8w)hN and (15w)hN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34(+) HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell-predominant humanized mice, HL was seen in T-cell-predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2(+), hCD20(+), hKi67(+), hCD20(+)/EBNA1(+), and EBER(+)) but negative for HL markers (LMP1(-), EBNA2(-), and hCD30(-)), most HL-like tumors were characterized by the presence of malignant Hodgkin's Reed-Sternberg (HRS)-like cells, lacunar RS (hCD30(+), hCD15(+), IgJ(-), EBER(+)/hCD30(+), EBNA1(+)/hCD30(+), LMP(+)/EBNA2(-), hCD68(+), hBCL2(-), hCD20(-/weak,) Phospho STAT6(+)), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.

Asunto(s)

Herpesvirus Humano 4/patogenicidad , Linfocitos/clasificación , Linfoma de Células B/inmunología , Linfoma de Células B/virología , Animales , Humanos , Linfoma de Células B/clasificación , Ratones , Ratones Endogámicos NOD , Ratones SCID

RESUMEN

The consistent presence of Epstein-Barr virus (EBV) in malignant cells of EBV-associated gastric carcinoma (EBVaGC) suggests it plays an important role during the development of EBVaGC. However, the entire genomic sequence of EBV from EBVaGC has yet to be determined. This study first determined, annotated, and analyzed the full genomic sequence of EBV from the naturally infected gastric carcinoma cell line SNU-719 using next-generation sequencing and comparative analyses. In consistent with the notion that EBV sequence isolates better reflect their geographic area than tissue origin, the SNU-719 EBV (named as GC1) was categorized as an East Asian type I EBV. Compared with the prototype B95.8 sequence, SNU-719 EBV contained 1372 variations, with 937 and 435 within coding and non-coding regions, respectively. Of the 937 variations, 465 were non-synonymous changes, while 472 synonymous changes included partial internal deletions in the coding regions of LMP1 and gp350. The RNAseq transcriptome revealed that multiple BART transcripts comprised the majority of EBV RNA reads. The SNU-719 EBV expressed high levels of BART, LF3, BHLF1, and BNLF2. Evidence of RNA editing at multiple sites in the host chromosome was found; however, no evidence of genome integration was seen. The annotated SNU-719 EBV sequence will be a useful reference in future EBVaGC studies.

Asunto(s)

Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Neoplasias Gástricas/virología , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Línea Celular Tumoral , Mapeo Cromosómico , Evolución Molecular , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ARN , Mutación Silenciosa , Factores de Transcripción , Transcriptoma , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genética

RESUMEN

Both the thymus (T) and bone (B) are necessary hematopoietic niches in adult humans. We previously showed that co-transplantation of human fetal T and B tissues into neonatal immunodeficient NOD/SCID IL2Rγ(null) (NSG, N) mice facilitated hematopoiesis. However, transplantation into neonatal mice resulted in high frequency of early death, making it unrealistic for repetitive experiments. In this study, young adult N mice were pre-engrafted with T and B, T alone, B alone or no tissues. The animals were irradiated and injected with autologous fetal liver (FL)-derived CD34(+) cells (34). The resultant mice were TB34N, T34N, B34N and 34N, respectively, and challenged with T cell dependent antigens (Ags). The humanized TB34N mice showed best performance of these mouse models in many aspects resembling the adult human Ag-experienced spleen. The TB34N mice exhibited better hematopoietic reconstitution; balanced development of T- and B-cell, and common progenitor cells; follicular lymphoid structures with a functional germinal center (GC) enriched with follicular dendritic cells (FDCs) and plasma cells (PCs); secretion of hIgG in the sera in response to Ags at comparable levels to those of human; derivations of hIgG mAb-secreting hybridoma clones. Collectively, the humanized TB34N mice could develop an adaptive immunity that was capable of producing Ag-specific hIgG at a significant level via class switching. This unprecedented TB34N platform in humanized mice would be useful in dissecting human immunity, for generating human Abs and clinical applications.

Asunto(s)

Inmunidad Adaptativa/inmunología , Anticuerpos/inmunología , Antígenos CD34/metabolismo , Trasplante Óseo , Trasplante de Tejido Fetal , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Bazo/inmunología , Timo/trasplante , Animales , Formación de Anticuerpos , Hematopoyesis , Xenoinjertos , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Bazo/patología

RESUMEN

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.

Asunto(s)

Genes Virales , Herpesvirus Humano 4/fisiología , Latencia del Virus , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Humanos , MicroARNs/genética , Neoplasias/etiología , Unión Proteica , ARN Viral/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo

RESUMEN

The spontaneous metastasis from human gastric carcinoma (GC) remains poorly reproduced in animal models. Here, we established an experimental mouse model in which GC progressively developed in the orthotopic stomach wall and metastasized to multiple organs; the tumors colonized in the ovary exhibited typical characteristics of Krukenberg tumor. The expression of mesenchymal markers was low in primary tumors and high in those in intravasating and extravasating veins. However, the expression of epithelial markers did not differ, indicating that the acquisition of mesenchymal markers without a concordant loss of typical epithelial markers was associated with metastasis. We identified 35 differentially expressed genes (DEGs) in GC cells metastasized to ovary, among which overexpression of GAGE12 family genes, the top-ranked DEGs, were validated. In addition, knockdown of the GAGE12 gene family affected transcription of many of the aforementioned 35 DEGs and inhibited trans-well migration, tumor sphere formation in vitro and tumor growth in vivo. In accordance, GAGE12 overexpression augmented migration, tumor sphere formation and sustained in vivo tumor growth. Taken together, the GAGE12 gene family promotes GC growth and metastasis by modulating the expression of GC metastasis-related genes.

Asunto(s)

Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Metástasis de la Neoplasia/genética , Neoplasias Gástricas/patología , Animales , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/secundario , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo

RESUMEN

BACKGROUND & AIMS: Patients with Epstein-Barr virus-associated gastric carcinoma (EBVaGC) have a better prognosis than those with gastric cancer not associated with EBV infection (EBVnGC). This is partly because EBV infection recruits lymphocytes, which infiltrate the tumor. A high degree of tumor heterogeneity is likely to be associated with poor response. We investigated differences in gene expression patterns between EBVaGC and EBVnGC. METHODS: We used gene expression profile analysis to compare tumor and nontumor gastric tissues from 12 patients with EBVaGC and 14 patients with EBVnGC. Findings were validated by whole transcriptome RNAseq and real-time quantitative polymerase chain reaction analyses. CD3(+) primary T cells were isolated from human blood samples; migration of these cells and of Jurkat cells were measured in culture with EBV-infected and uninfected gastric cancer cells. RESULTS: Based on Pearson correlation matrix analysis, EBVaGCs had a higher degree of homogeneity than EBVnGCs. Although 4550 genes were differentially expressed between tumor and nontumor gastric tissues of patients with EBVnGC, only 186 genes were differentially expressed between tumor and nontumor gastric tissues of patients with EBVaGC (P < .001). This finding supports the concept that EBVaGCs have fewer genetic and epigenetic alterations than EBVnGCs. Expression of major histocompatibility complex class II genes and genes that regulate chemokine activity were more often deregulated in EBVaGCs compared with nontumor tissues. In culture, more T cells migrated to EBV-infected gastric cancer cells than to uninfected cells; migration was blocked with a neutralizing antibody against CXCR3 (a receptor for many chemokines). CONCLUSIONS: Fewer genes are deregulated in EBVaGC than in EBVnGC. Most changes in EBVaGCs occur in immune response genes. These changes might allow EBVaGC to recruit reactive immune cells; this might contribute to the better outcomes of these patients compared with those with EBVnGC.

Asunto(s)

Carcinoma/genética , Carcinoma/inmunología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias Gástricas/genética , Neoplasias Gástricas/inmunología , Linfocitos T/inmunología , Anticuerpos Neutralizantes/farmacología , Carcinoma/virología , Estudios de Casos y Controles , Quimiotaxis de Leucocito , Análisis por Conglomerados , Técnicas de Cocultivo , Infecciones por Virus de Epstein-Barr/complicaciones , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Humanos , Células Jurkat , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR3/antagonistas & inhibidores , Receptores CXCR3/inmunología , Análisis de Secuencia de ARN , Neoplasias Gástricas/virología , Linfocitos T/efectos de los fármacos , Linfocitos T/virología , Células Tumorales Cultivadas

RESUMEN

CD10 expression was identified as a contributor to cancer progression in several cancers; however, the exact biological significance and mechanism of CD10 expression remains unclear. In addition, CD10 expression in esophageal squamous cell carcinoma (ESCC) has not been studied. We investigated the relationship between CD10 and Twist1. Furthermore, we examined the effect of CD10 on tumorigenicity using in vivo and in vitro systems as well as establishing the clinical significance of CD10 expression in ESCC using large clinical samples. CD10 expression was upregulated by Twist1 and there was a strong correlation between mRNA and protein expression. Twist1 can specifically upregulate CD10 at the transcriptional level via an interaction with the promoter region of CD10 and the proximal E-box CAGGTG in the CD10 promoter was identified as a binding site for Twist1. CD10 is frequently expressed in ESCC cell lines and silencing CD10 suppresses migration/invasion and anchorage-independent tumor growth of ESCC cells. Knockdown of CD10 inhibits the growth of ESCC xenograft in nude mice, suggesting that CD10 plays a role in enhancing the tumorigenesis of ESCC. From among 153 ESCC samples, 46 (30.0%) showed varying degrees of CD10 expression in cancer cells. In addition, stromal fibroblasts also showed varying amounts of CD10 expression in 92 (60.9%) tumor samples. CD10 overexpression in cancer cells as well as in stromal fibroblasts was an independent poor prognostic factor in both overall survival and disease-free survival. CD10 could be a promising target for the treatment of ESCC.

Asunto(s)

Carcinoma de Células Escamosas/patología , Movimiento Celular , Proliferación Celular , Neoplasias Esofágicas/patología , Neprilisina/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Anciano , Animales , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Adhesión Celular , Inmunoprecipitación de Cromatina , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Estadificación de Neoplasias , Neprilisina/genética , Proteínas Nucleares/genética , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismo , Células del Estroma/patología , Tasa de Supervivencia , Células Tumorales Cultivadas , Proteína 1 Relacionada con Twist/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

BACKGROUND: Circulating tumor cells (CTCs) can be used to predict the spread of cancer to distant sites, to monitor the clinical response to therapy and to predict patient survival. The currently used EpCAM antibody-mediated identification of CTCs may lead to false negative results due to the low level or absence of EpCAM expression in several types of cancer, thus provoking a need to identify novel CTC markers. METHODS: The Cancer Cell Line Encyclopedia (CCLE) microarray dataset, storing 18,915 gene expression profiles across 967 cancer cell lines derived from 25 primary sites, was systematically analyzed. The results obtained were cross-validated using an independent microarray dataset generated from 1,911 clinical cancer specimens derived from 15 different cancers. RESULTS: Through bioinformatics analyses we identified, categorized and prioritized three classes of novel markers: pan-CTC markers (n = 45), EpCAM((-/low)) CTC markers (n = 16) and single cancer type-specific markers (n = 74). The pan-CTC markers were significantly, uniformly and constitutively over-expressed in most cancer types, except in cancers of hematopoietic and lymphoid origin. The EpCAM((-/low)) CTC markers were over-expressed in cancers with low or undetectable EpCAM expression levels. Among these, 22 markers were validated in an independent microarray dataset. In addition, 74 markers that were over-expressed in only single cancer types were categorized. CONCLUSIONS: The combined use of these novel markers in conjunction with cancer type-specific markers should be able to quantify CTCs that are not captured by EpCAM antibodies, and to enhance the sensitivity and specificity of CTC detection among admixtures containing leucocytes or other types of contaminants.

Asunto(s)

Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Neoplásicas Circulantes/metabolismo , Anticuerpos/análisis , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Biología Computacional/métodos , Bases de Datos Genéticas , Molécula de Adhesión Celular Epitelial , Humanos

RESUMEN

The replication and persistence of extra chromosomal Epstein-Barr virus (EBV) episome in latently infected cells are primarily dependent on the binding of EBV-encoded nuclear antigen 1 (EBNA1) to the cognate EBV oriP element. In continuation of the previous study, herein we characterized EBNA1 small molecule inhibitors (H20, H31) and their underlying inhibitory mechanisms. In silico docking analyses predicted that H20 fits into a pocket in the EBNA1 DNA binding domain (DBD). However, H20 did not significantly affect EBNA1 binding to its cognate sequence. A limited structure-relationship study of H20 identified a hydrophobic compound H31, as an EBNA1 inhibitor. An in vitro EBNA1 EMSA and in vivo EGFP-EBNA1 confocal microscopy analysis showed that H31 inhibited EBNA1-dependent oriP sequence-specific DNA binding activity, but not sequence-nonspecific chromosomal association. Consistent with this, H31 repressed the EBNA1-dependent transcription, replication, and persistence of an EBV oriP plasmid. Furthermore, H31 induced progressive loss of EBV episome. In addition, H31 selectively retarded the growth of EBV-infected LCL or Burkitt's lymphoma cells. These data indicate that H31 inhibition of EBNA1-dependent DNA binding decreases transcription from and persistence of EBV episome in EBV-infected cells. These new compounds might be useful probes for dissecting EBNA1 functions in vitro and in vivo.

Asunto(s)

Antivirales/farmacología , Replicación del ADN/efectos de los fármacos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Genoma Viral , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Replicación Viral/efectos de los fármacos , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos

RESUMEN

PURPOSE: Metastatic relapse of primary lung cancer leads to therapeutic resistance and unfavorable clinical prognosis; therefore, identification of key molecules associated with metastatic conversion has significant clinical implications. We previously identified a link between early brain metastasis of lung adenocarcinoma and amplification of the α-smooth muscle actin (ACTA2) gene. The aim of present study was to investigate the prognostic and functional significance of ACTA2 expression in cancer cells for the metastatic potential of lung adenocarcinomas. EXPERIMENTAL DESIGN: ACTA2 expression was analyzed in tumor cells from 263 patients with primary lung adenocarcinomas by immunohistochemistry, and was correlated with clinicopathologic parameters. The expression of ACTA2 in human lung adenocarcinoma cells was modulated with short hairpin RNAs (shRNA) and siRNAs specifically targeting ACTA2. RESULTS: The patients with lung adenocarcinomas with high ACTA2 expression in tumor cells showed significantly enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of lung adenocarcinoma cells without affecting proliferation. Consistent with the in vitro results, depletion of ACTA2 in human lung adenocarcinoma PC14PE6 cells significantly reduced their metastatic potential without altering their tumorigenic potential. Expression of c-MET and FAK in lung adenocarcinoma cells was also reduced by ACTA2-targeting siRNAs and shRNAs, and was accompanied by a loss of mesenchymal characteristics. CONCLUSIONS: These findings indicate that ACTA2 regulates c-MET and FAK expression in lung adenocarcinoma cells, which positively and selectively influence metastatic potential. Therefore, ACTA2 could be a promising prognostic biomarker and/or therapeutic target for metastatic lung adenocarcinoma.

Asunto(s)

Actinas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Actinas/genética , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Ratones , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-met/metabolismo , Interferencia de ARN

RESUMEN

Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.

Asunto(s)

Linfocitos B/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Latencia del Virus/fisiología , Linfocitos B/virología , Línea Celular Tumoral , Antígenos Nucleares del Virus de Epstein-Barr/genética , Humanos , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Nucleofosmina , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética , Proteínas Virales/genética , Ensamble de Virus/fisiología

RESUMEN

Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two novel inhibitors of EBNA1 dimerization. This study demonstrates that EBNA1 homodimerization can be effectively targeted by a small molecule or peptide.

Asunto(s)

Antivirales/farmacología , Benzoatos/farmacología , Proteínas de Unión al ADN/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Multimerización de Proteína/efectos de los fármacos , Tiobarbitúricos/farmacología , Antivirales/química , Benzoatos/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Antígenos Nucleares del Virus de Epstein-Barr/química , Humanos , Péptidos/química , Péptidos/farmacología , Origen de Réplica , Tiobarbitúricos/química