RESUMEN
BACKGROUND: Cancer-associated fibroblasts (CAFs), integral to the tumour microenvironment, are pivotal in cancer progression, exhibiting either pro-tumourigenic or anti-tumourigenic functions. Their inherent phenotypic and functional diversity allows for the subdivision of CAFs into various subpopulations. While several classification systems have been suggested for different cancer types, a unified molecular classification of CAFs on a single-cell pan-cancer scale has yet to be established. METHODS: We employed a comprehensive single-cell transcriptomic atlas encompassing 12 solid tumour types. Our objective was to establish a novel molecular classification and to elucidate the evolutionary trajectories of CAFs. We investigated the functional profiles of each CAF subtype using Single-Cell Regulatory Network Inference and Clustering and single-cell gene set enrichment analysis. The clinical relevance of these subtypes was assessed through survival curve analysis. Concurrently, we employed multiplex immunofluorescence staining on tumour tissues to determine the dynamic changes of CAF subtypes across different tumour stages. Additionally, we identified the small molecule procyanidin C1 (PCC1) as a target for matrix-producing CAF (matCAF) using molecular docking techniques and further validated these findings through in vitro and in vivo experiments. RESULTS: In our investigation of solid tumours, we identified four molecular clusters of CAFs: progenitor CAF (proCAF), inflammatory CAF (iCAF), myofibroblastic CAF (myCAF) and matCAF, each characterised by distinct molecular traits. This classification was consistently applicable across all nine studied solid tumour types. These CAF subtypes displayed unique evolutionary pathways, functional roles and clinical relevance in various solid tumours. Notably, the matCAF subtype was associated with poorer prognoses in several cancer types. The targeting of matCAF using the identified small molecule, PCC1, demonstrated promising antitumour activity. CONCLUSIONS: Collectively, the various subtypes of CAFs, particularly matCAF, are crucial in the initiation and progression of cancer. Focusing therapeutic strategies on targeting matCAF in solid tumours holds significant potential for cancer treatment.
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Fibroblastos Asociados al Cáncer , Neoplasias , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Simulación del Acoplamiento Molecular , Neoplasias/patología , Perfilación de la Expresión Génica , Transcriptoma/genética , Microambiente Tumoral/genéticaRESUMEN
Serrated polyps are a clinically and molecularly heterogeneous group of lesions that can contribute to the development of colorectal cancers (CRCs). However, the molecular mechanism underlying the development of serrated lesions is still not well understood. Here, we combined multiple approaches to analyze the genetic alterations in 86 colorectal adenomas (including 35 sessile serrated lesions, 15 traditional adenomas, and 36 conventional adenomatous polyps). We also investigated the in vitro and in vivo oncogenic properties of a novel variant of the NCOA4-RET fusion gene. Molecular profiling revealed that sessile serrated lesions and traditional serrated adenomas have distinct clinicopathological and molecular features. Moreover, we identified receptor tyrosine kinase translocations exclusively in sessile serrated lesions (17%), and the observation was validated in a separate cohort of 34 sessile serrated lesions (15%). The kinase fusions as well as the BRAF and KRAS mutations were mutually exclusive to each other. Ectopic expression of a novel variant of the NCOA4-RET fusion gene promoted cell proliferation in vitro and in vivo, and the proliferation was significantly suppressed by RET kinase inhibitors. All of these underscored the importance of mitogen-activated protein kinase (MAPK) pathway activation in the serrated pathway of colorectal tumorigenesis. In addition, we demonstrated that the kinase fusion may occur early in the precursor lesion and subsequent loss of TP53 may drives the transformation to carcinoma during serrated tumorigenesis. In conclusion, we identified kinase fusions as a significant alternative driver of the serrated pathway in colorectal cancer development, and detecting their presence may serve as a biomarker for the diagnosis of sessile serrated lesions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Neoplasias del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Proteínas Tirosina Quinasas Receptoras/genética , Adenoma/genética , Adenoma/patología , Animales , Neoplasias del Colon/genética , Humanos , Hiperplasia/genética , Hiperplasia/patología , Ratones , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Colorectal cancer (CRC) is a heterogeneous disease with complex mechanisms of pathogenesis. Classification systems have been proposed based on molecular features of tumors in clinical practice. Thus, more molecular markers associated with development and progression of CRC might serve as useful tools for early diagnosis even for providing more accurate molecular classification. Frequent gain of chromosome 8q was detected in CRC by array-CGH and overexpression of exosome component 4 (EXOSC4) in this region was revealed by expression microarray analysis. Through qRT-PCR and immunohistochemistry (IHC) analysis, EXOSC4 showed increased expression in CRC cell lines and clinical specimens. Higher expression of EXOSC4 was more often detected in left side, and correlated with BRAF wild type, MSI-low or MSS, CIMP-low, and MLH1-no-silence CRC patients. Functionally, EXOSC4 overexpression increased early tumorigenic capacity by promoting cell proliferation and monolayer colony formation, enhancing cell invasion and migration study and accelerating xenograft formation in nude mice. While EXOSC4 knockdown exhibited anti-oncogenic role such as inhibiting cell proliferation and invasion. EXOSC4 inhibition also resulted in G1 phase cell cycle arrest. For the downstream signaling analysis, EXOSC4 was found to be involved in multiple signaling pathways such as cell cycle, p53 pathway and Wnt pathway. In summary, our findings demonstrated the oncogenic role of EXOSC4 in development and progression of CRC. Deep understanding of EXOSC4 as a potential diagnostic molecular biomarker will provide clinical translational potential for intervention therapy.
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Neoplasias Colorrectales/patología , Variaciones en el Número de Copia de ADN , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Regulación hacia Arriba , Animales , Células CACO-2 , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Hibridación Genómica Comparativa , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HT29 , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Transducción de SeñalRESUMEN
Immune checkpoint blockade targeting the PD-1/PD-L1 axis has recently demonstrated efficacy and promise in cancer treatment. Appropriate biomarker selection is therefore essential for improving treatment efficacy. However, the establishment of PD-L1 assay in pathology laboratories is complicated by the presence of multiple testing platforms using different scoring systems. Here we assessed the PD-L1 expression in 713 consecutive non-small cell lung carcinomas by four commercially available PD-L1 immunohistochemical assays, namely, 22C3, 28-8, SP142 and SP263. The analytical performances of the four assays and diagnostic performances across clinically relevant cutoffs were evaluated. The prevalence of PD-L1 (22C3) expression was 21% with a ≥50% cutoff and 56% with a ≥1% cutoff. High PD-L1 expression (using a ≥50% cutoff) was significantly associated with male sex (P = 0.001), ever smoking history (P < 0.001), squamous cell carcinoma (P = 0.001), large cell carcinoma (P < 0.001), lymphoepithelioma-like carcinoma (P = 0.006), sarcomatoid carcinoma (P < 0.001), mutant KRAS (P = 0.005) and wild-type EGFR (P = 0.003). Elevated PD-L1 expression was also significantly associated with shorter survival in patients with adenocarcinoma (log-rank P = 0.026) and remained an independent prognostic factor by multivariable analysis. Among the four assays, 22C3, 28-8 and SP263 were highly concordant for tumor cell scoring. With a cutoff of ≥50% (i.e., the threshold for first-line patient selection), inter-rater agreement was high among the three assays with percentage agreement >97%. In conclusion, three PD-L1 assays showed good analytical performance and a high agreement with each other, but not all cases were correctly classified using the same clinical cutoff. Further studies comparing the predictive value of these assays are required to address the interchangeability of these assays for clinical use.
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Antígeno B7-H1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Femenino , Humanos , Inmunohistoquímica , Pulmón/patología , Pulmón/cirugía , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de SupervivenciaRESUMEN
miR-375 is a tumor-suppressive microRNA (miRNA) in gastric cancer (GC). However, its molecular mechanism remains unclear. The aim of this study is to comprehensively investigate how miR-375 is involved in Hippo pathway by targeting multiple oncogenes. miR-375 expression in gastric cancer cell lines and primary GC was investigated by qRT-PCR. The regulation of YAP1, TEAD4, and CTGF expression by miR-375 was evaluated by qRT-PCR, western blot, and luciferase reporter assays, respectively. The functional roles of the related genes were examined by siRNA-mediated knockdown or ectopic expression assays. The clinical significance and expression correlation analysis of miR-375, YAP1, and CTGF were performed in primary GCs. TCGA cohort was also used to analyze the expression correlation of YAP1, TEAD4, CTGF, and miR-375 in primary GCs. miR-375 was down-regulated in GC due to promoter methylation and histone deacetylation. miR-375 downregulation was associated with unfavorable outcome and lymph node metastasis. Ectopic expression of miR-375 inhibited tumor growth in vitro and in vivo. Three components of Hippo pathway, YAP1, TEAD4 and CTGF, were revealed to be direct targets of miR-375. The expression of three genes showed a negative correlation with miR-375 expression and YAP1 re-expression partly abolished the tumor-suppressive effect of miR-375. Furthermore, CTGF was confirmed to be the key downstream of Hippo-YAP1 cascade and its knockdown phenocopied siYAP1 or miR-375 overexpression. YAP1 nuclear accumulation was positively correlated with CTGF cytoplasmic expression in primary GC tissues. Verteporfin exerted an anti-oncogenic effect in GC cell lines by quenching CTGF expression through YAP1 degradation. In short, miR-375 was involved in the Hippo pathway by targeting YAP1-TEAD4-CTGF axis and enriched our knowledge on the miRNA dysregulation in gastric tumorigenesis.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinogénesis/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Proteínas de Unión al ADN/metabolismo , MicroARNs/metabolismo , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Neoplasias Gástricas/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Vía de Señalización Hippo , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Neoplasias Gástricas/patología , Factores de Transcripción de Dominio TEA , Proteínas Señalizadoras YAPRESUMEN
Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , MicroARNs/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , ARN Viral/genética , Regiones no Traducidas 3' , Animales , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Sitios de Unión , Línea Celular Tumoral , Daño del ADN , Represión Enzimática , Infecciones por Virus de Epstein-Barr/diagnóstico , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Carcinoma Nasofaríngeo/enzimología , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/enzimología , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virología , Transcriptoma , Latencia del VirusRESUMEN
INTRODUCTION: Oncogenic driver mutations activating receptor tyrosine kinase pathways are promising predictive markers for targeted treatment. We investigated the mutation profile of an updated driver events list on receptor tyrosine kinase/RAS/PI3K axis and the clinicopathologic implications in a cohort of never-smoker predominated Chinese lung adenocarcinoma. METHODS: We tested 154 lung adenocarcinomas and adenosquamous carcinomas for EGFR, KRAS, HER2, BRAF, PIK3CA, MET, NRAS, MAP2K1, and RIT1 mutations by polymerase chain reaction-direct sequencing. MET amplification and ALK and ROS1 translocations were assessed by fluorescent in situ hybridizations. MET and thyroid transcription factor-1 protein expressions were investigated by immunohistochemistry. RESULTS: Seventy percent of lung adenocarcinomas carried actionable driver events. Alterations on EGFR (43%), KRAS (11.4%), ALK (6%), and MET (5.4%) were frequently found. ROS1 translocation and mutations involving BRAF, HER2, NRAS, and PIK3CA were also detected. No mutation was observed in RIT1 and MAP2K1. Patients with EGFR mutations had a favorable prognosis, whereas those with MET mutations had poorer overall survival. Multivariate analysis further demonstrated that MET mutation was an independent prognostic factor. Although MET protein expression was detected in 65% of lung adenocarcinoma, only 10% of the MET-immunohistochemistry positive tumors harbor MET DNA alterations that drove protein overexpression. Appropriate predictive biomarker is essential for selecting patients who might benefit from specific targeted therapy. CONCLUSION: Actionable driver events can be detected in two thirds of lung adenocarcinoma. MET DNA alterations define a subset of patients with aggressive diseases that might potentially benefit from anti-MET targeted therapy. High negative predictive values of thyroid transcription factor-1 and MET expression suggest potential roles as surrogate markers for EGFR and/or MET mutations.
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Adenocarcinoma/genética , Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-met/genética , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-met/metabolismo , Estudios RetrospectivosRESUMEN
BACKGROUND: MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated. METHODS: The expression of miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated. RESULTS: We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression. CONCLUSION: In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.
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Proteínas Adaptadoras Transductoras de Señales/genética , Adenocarcinoma/genética , Genes Supresores de Tumor/fisiología , MicroARNs/genética , Fosfoproteínas/genética , Neoplasias Gástricas/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo/genética , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fase de Descanso del Ciclo Celular/genética , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: Aberrant AKT activation contributes to gastric cancer cell survival and chemotherapy resistance, however its regulation is poorly understood. microRNAs have been established to be important regulators in gastric carcinogenesis. Here, we showed the functional role and putative target of let-7b and let-7g (let-7b/g) in gastric carcinogenesis. METHODS: The expression of let-7b/g in gastric cancer cell lines and primary tumors were evaluated by miRNA qRT-PCR. The putative target gene of let-7b/g was explored by TargetScan followed by further validation. Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches. RESULTS: let-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3'UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6. AKT2 mRNA expression showed negative correlation with the expression of let-7b/g in primary tumors. Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g. Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g. CONCLUSION: In conclusion, our findings reveal decreased let-7b/g contributes to aberrant AKT activation in gastric tumorigenesis and provide a potential therapeutic strategy for gastric cancer.
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Carcinogénesis/genética , Carcinogénesis/patología , Silenciador del Gen , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones Desnudos , Datos de Secuencia Molecular , Unión Proteica/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Análisis de Supervivencia , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer are unclear. We investigated EBV-associated genomic and epigenomic variations in gastric cancer cells and tumors. METHODS: We performed whole-genome, transcriptome, and epigenome sequence analyses of a gastric adenocarcinoma cell line (AGS cells), before and after EBV infection. We then looked for alterations in gastric tumor samples, with (n = 34) or without (n = 100) EBV infection, collected from patients at the Prince of Wales Hospital, Chinese University of Hong Kong (from 1998 through 2004), or the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (from 1999 through 2006). RESULTS: Transcriptome analysis showed that infected cells expressed 9 EBV genes previously detected in EBV-associated gastric tumors and 71 EBV genes not previously reported in gastric tumors. Ten viral genes that had not been reported previously in gastric cancer but were expressed most highly in EBV-infected cells also were expressed in primary EBV-positive gastric tumors. Whole-genome sequence analysis identified 45 EBV-associated nonsynonymous mutations. These mutations, in genes such as AKT2, CCNA1, MAP3K4, and TGFBR1, were associated significantly with EBV-positive gastric tumors, compared with EBV-negative tumors. An activating mutation in AKT2 was associated with reduced survival times of patients with EBV-positive gastric cancer (P = .006); this mutation was found to dysregulate mitogen-activated protein kinase signaling. Integrated epigenome and transcriptome analyses identified 216 genes transcriptionally down-regulated by EBV-associated hypermethylation; methylation of ACSS1, FAM3B, IHH, and TRABD increased significantly in EBV-positive tumors. Overexpression of Indian hedgehog (IHH) and TraB domain containing (TRABD) increased proliferation and colony formation of gastric cancer cells, whereas knockdown of these genes reduced these activities. We found 5 signaling pathways (axon guidance, focal adhesion formation, interactions among cytokines and receptors, mitogen-activated protein kinase signaling, and actin cytoskeleton regulation) to be affected commonly by EBV-associated genomic and epigenomic alterations. CONCLUSIONS: By using genomic, transcriptome, and epigenomic comparisons of EBV infected vs noninfected gastric cancer cells and tumor samples, we identified alterations in genes, gene expression, and methylation that affect different signaling networks. These might be involved in EBV-associated gastric carcinogenesis.
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Adenocarcinoma/genética , Infecciones por Virus de Epstein-Barr/genética , Estudio de Asociación del Genoma Completo , Herpesvirus Humano 4/genética , Neoplasias Gástricas/genética , Transcriptoma , Adenocarcinoma/virología , Línea Celular Tumoral , Ciclina A1/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Infecciones por Virus de Epstein-Barr/virología , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Genes Virales , Humanos , MAP Quinasa Quinasa Quinasa 4/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Neoplasias Gástricas/virologíaRESUMEN
The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects ß-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.
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Carcinoma Hepatocelular/genética , Proteínas de Unión al ADN/genética , Neoplasias Hepáticas/genética , Transactivadores/genética , Proteínas Virales de Fusión/genética , Animales , Secuencia de Bases , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Movimiento Celular/genética , Dietilnitrosamina , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica , Virus de la Hepatitis B/genética , Humanos , Neoplasias Hepáticas/virología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de ADN , Transcripción Genética/genética , Activación Transcripcional/genética , Transcriptoma/genética , Proteínas Reguladoras y Accesorias Virales , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P<0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10-16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.
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Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Adenocarcinoma/epidemiología , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Secuencia de Bases , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Células HEK293 , Humanos , Mutación INDEL , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Mutación Missense , Células 3T3 NIH , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Distribución por Sexo , Transducción de Señal , Adulto JovenRESUMEN
INTRODUCTION: The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as a potent oncogenic driver in non-small-cell lung cancer, in particular adenocarcinoma (ADC). It defines a unique subgroup of lung ADC, which may be responsive to ALK inhibitors. Detection of ALK rearrangement by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) is considered to be the standard procedure, but each with its own limitation. We evaluated the practical usefulness of immunohistochemistry (IHC) to detect ALK expression as a reliable detection method of ALK rearrangement in lung ADC. METHODS: We tested 373 lung ADCs for ALK rearrangement by IHC and FISH. Multiplex RT-PCR was performed to confirm the fusion variants. RESULTS: Twenty-two of 373 lung ACs (5.9%) were positive for ALK immunoreactivity. ALK-positive tumor cells demonstrated strong and diffused granular staining in the cytoplasm. All the ALK IHC-positive cases were confirmed to harbor ALK rearrangement, either by FISH, or RT-PCR. Two cases with positive ALK protein expression, but negative for breakapart FISH signal were shown to harbor EML4-ALK variant 1 by RT-PCR. None of the ALK IHC-negative cases were FISH-positive. In addition, we identified a novel EML4-ALK fusion variant (E3:ins53A20), and its potent transformation potential has been confirmed by in vivo tumorigenicity assay. CONCLUSION: IHC can effectively detect ALK rearrangement in lung cancer. It might provide a reliable and cost-effective diagnostic approach in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy.
Asunto(s)
Adenocarcinoma/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Reordenamiento Génico , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas Receptoras/genética , Adenocarcinoma/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico , Animales , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Transformación Celular Neoplásica , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Variación Genética , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/diagnóstico , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Células 3T3 NIH , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & AIMS: Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. METHODS: Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n=73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. RESULTS: The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients (p<0.009). CONCLUSIONS: Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.
Asunto(s)
Carcinoma Hepatocelular/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Hepáticas/genética , ARN Interferente Pequeño/genética , ARN no Traducido/genética , Transcriptoma , Adulto , Anciano , Carcinoma Hepatocelular/mortalidad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/mortalidad , Masculino , MicroARNs/fisiología , Persona de Mediana EdadRESUMEN
BACKGROUND: Aberrant methylation of tumor-related genes has been reported in Epstein-Barr virus (EBV)-associated gastric cancers. This study sought to profile EBV-driven hypermethylation in EBV-infected cells. METHODS: The EBV-positive AGS gastric cancer cell line (AGS-EBV) and EBV-negative AGS cells were used in this study. DNA methyltransferase-3b (DNMT3b) activity was assessed by EpiQuick activity assay, and genome-wide DNA methylation profiles were assessed by methyl-DNA immunoprecipitation microarray assay. RESULTS: EBV infection was confirmed in AGS-EBV cells by EBV-encoded RNA in situ hybridization. Expression and activity of DNA methyltransferase-3b (DNMT3b) was significantly increased in AGS-EBV compared to AGS. Ectopic expression of LMP2A (latent membrane protein 2A) in AGS increased activity of DNMT3b. A total of 1065 genes were differentially methylated by EBV infection (fold-changes ≥ 2, P < .05) in AGS-EBV compared to AGS cells. The majority of the differentially methylated genes (83.2%, 886 of 1065 genes) had cytosine-guanine dinucleotide (CpG) hypermethylation in AGS-EBV (fold-changes 2.43â¼65.2) versus that found in AGS cells. Gene ontology analysis revealed that hypermethylated genes were enriched in the important cancer pathways (≥ 10 genes each, P ≤ .05) including mitogen-activated protein kinase signaling, cell adhesion molecules, wnt signaling pathway, and so forth. Six novel hypermethylated candidates (IL15RA, REC8, SSTR1, EPHB6, MDGA2, and SCARF2) were further validated. Higher levels of DNA methylation were confirmed for all these genes in AGS-EBV cells by bisulfite genomic sequencing. Furthermore, these candidates were silenced or down-regulated in AGS-EBV cells, but can be restored by demethylation treatment. CONCLUSIONS: EBV infection in AGS cells induced aberrant CpG hypermethylation of 886 genes involving in important cancer-related pathways. Induction of promoter methylation by EBV is regulated by up-regulation of DNMT3b through LMP2A.
Asunto(s)
Metilación de ADN , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Neoplasias Gástricas/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Activación Enzimática , Epigénesis Genética , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Interacciones Huésped-Patógeno , Humanos , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de la Familia Eph , Receptores de Interleucina-15/genética , Receptores de Somatostatina/genética , Análisis de Secuencia de ADN , Neoplasias Gástricas/virología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , ADN Metiltransferasa 3BRESUMEN
Stathmin1 (STMN1) is a candidate oncoprotein and prognosis marker in several kinds of cancers. This study was aimed to analyze its expression and biological functions in gastric cancer. The expression of STMN1 was evaluated by qRT-PCR, western blot and immunohistochemistry. The biological function of STMN1 was determined by MTT proliferation assays, monolayer colony formation and cell invasion assays using small interference RNA technique in gastric cancer cell lines. We also explored the regulation of STMN1 expression by microRNA-223. STMN1 was upregulated in gastric cancer cell lines and primary gastric adenocarcinomas. STMN1-positive tumors were more likely to be found in old age group and associated with p53 nuclear expression. In diffuse type gastric adenocarcinomas, STMN1 expression was correlated with age (pâ=â0.043), T stage (pâ=â0.004) and lymph node metastasis (pâ=â0.046). Expression of STMN1 in diffuse type gastric adenocarcinoma was associated with poor disease specific survival by univariate analysis (pâ=â0.01). STMN1 knockdown in AGS and MKN7 cell lines suppressed proliferation (p<0.001), reduced monolayer colony formation (p<0.001), inhibited cell invasion and migration ability (p<0.001) and induced G1 phase arrest. siSTMN1 could also suppress cell growth in vivo (p<0. 01). We finally confirmed that STMN1 is a putative downstream target of miR-223 in gastric cancer. Our findings supported an oncogenic role of STMN1 in gastric cancer. STMN1 might serve as a prognostic marker and a potential therapeutic target for gastric cancer.
Asunto(s)
Adenocarcinoma/metabolismo , MicroARNs/metabolismo , Estatmina/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Anciano , Línea Celular Tumoral , Movimiento Celular , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Estatmina/antagonistas & inhibidores , Estatmina/genética , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patologíaRESUMEN
PURPOSE: Yes-associated protein 1 (YAP1) is a multifunctional protein that can interact with different transcription factors to activate gene expression. The role of YAP1 in tumorigenesis is unclear. We aimed to investigate the functional role of YAP1 in tumorigenesis of gastric cancer. EXPERIMENTAL DESIGN: YAP1 expression in gastric adenocarcinoma was evaluated. The biological function was determined by proliferation assay, colony formation, cell invasion, and flow cytometric analysis through knocking down or ectopic expressing YAP1 in gastric cancer cell lines coupled with in vivo study. The possible downstream effectors of YAP1 were investigated by expression microarray. RESULTS: YAP1 protein expression was upregulated in gastric cancer. Nuclear accumulation of YAP1 was associated with poor disease-specific survival (P = 0.021), especially in patients with early-stage diseases (P < 0.001). Knockdown YAP1 resulted in a significant reduction in proliferation, anchorage-dependent colony formation, cell invasion, and cell motility. Ectopic YAP1 expression promoted anchorage-independent colony formation, induced a more invasive phenotype, and accelerated cell growth both in vitro and in vivo. Microarray analysis highlighted the alteration of MAPK (mitogen-activated protein kinase) pathway by YAP1. We confirmed a constitutive activation of RAF/MEK/ERK (extracellular signal-regulated kinase) in YAP1-expressing MKN45 cells and further showed that YAP1 enhanced serum/epidermal growth factor-induced c-Fos expression in gastric cancer cells. CONCLUSIONS: Our findings supported that YAP1 exhibits oncogenic property in gastric cancer. We provided the first evidence that YAP1 exerted the oncogenic function by enhancing the capacity to activate the early-response gene pathway. YAP1 could be a prognostic biomarker and potential therapeutic target for gastric cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Oncogenes/genética , Fosfoproteínas/genética , Pronóstico , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND & AIMS: Recent studies have emphasized causative links between microRNA (miRNA) deregulations and cancer development. In hepatocellular carcinoma (HCC), information on differentially expressed miRNA remained largely undefined. METHODS: Array-based miRNA profiling was performed on HCC cells that were derived from chronic carriers of hepatitis B virus (HBV) and hepatitis C virus (HCV), and nonviral-associated patients. Specific microRNA (miR)-223 and miR-222 deregulations were verified in an independent series of tumors. The functional effect of miR-223 was examined further. An integrative analysis of messenger RNA (mRNA) array with in silico predictions defined potential downstream targets of miR-223. A luciferase reporter assay was conducted to confirm target association. RESULTS: Distinct up-regulations of miR-222, miR-221, and miR-31, and down-regulations of miR-223, miR-126, and miR-122a were identified. Further investigations suggested the highly deregulated miR-223 and miR-222 could unequivocally distinguish HCC from adjacent nontumoral liver, irrespective of viral associations (P Asunto(s)
Carcinoma Hepatocelular/genética
, Regulación Neoplásica de la Expresión Génica
, Neoplasias Hepáticas/genética
, MicroARNs/genética
, Estatmina/genética
, Adulto
, Anciano
, Carcinoma Hepatocelular/virología
, Regulación hacia Abajo
, Femenino
, Hepatitis B Crónica/complicaciones
, Hepatitis C Crónica/complicaciones
, Humanos
, Neoplasias Hepáticas/virología
, Luciferasas/genética
, Masculino
, Persona de Mediana Edad
, Análisis de Secuencia por Matrices de Oligonucleótidos
RESUMEN
Epstein-Barr virus (EBV) infects nearly 90% of adults worldwide and is the pathogenic source of a broad spectrum of malignancies originating from lymphoid and epithelial cells. Currently, no vaccine has been developed to immunologically inactivate this virus. In infected patients, anti-EBV viral capsid antigen (VCA) immunoglobins represent some of the useful diagnostic markers for carcinoma development. To demonstrate that the EBV VCA antigen can be produced in plants, the plastid genome of tobacco (Nicotiana tabacum cv. SR1) was transformed with a VCA-expressing cassette. The EBV VCA mRNA was actively transcribed in the transplastomic plants and antigen production was detected. This study indicates that plastid transformation could be a promising strategy in EBV VCA antigen production.