RESUMO
Incomplete understanding of the metastatic process hinders personalized therapy. Here we report the most comprehensive whole-genome study of colorectal metastases vs. matched primary tumors. 65% of somatic mutations originate from a common progenitor, with 15% being tumor- and 19% metastasis-specific, implicating a higher mutation rate in metastases. Tumor- and metastasis-specific mutations harbor elevated levels of BRCAness. We confirm multistage progression with new components ARHGEF7/ARHGEF33. Recurrently mutated non-coding elements include ncRNAs RP11-594N15.3, AC010091, SNHG14, 3' UTRs of FOXP2, DACH2, TRPM3, XKR4, ANO5, CBL, CBLB, the latter four potentially dual protagonists in metastasis and efferocytosis-/PD-L1 mediated immunosuppression. Actionable metastasis-specific lesions include FAT1, FGF1, BRCA2, KDR, and AKT2-, AKT3-, and PDGFRA-3' UTRs. Metastasis specific mutations are enriched in PI3K-Akt signaling, cell adhesion, ECM and hepatic stellate activation genes, suggesting genetic programs for site-specific colonization. Our results put forward hypotheses on tumor and metastasis evolution, and evidence for metastasis-specific events relevant for personalized therapy.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Neoplasias Hepáticas/genética , Medicina de Precisão , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/secundário , Idoso , Anoctaminas/genética , Proteínas Reguladoras de Apoptose , Proteína BRCA2/genética , Adesão Celular/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Proteínas de Ligação a DNA , Matriz Extracelular/genética , Feminino , Fatores de Transcrição Forkhead/genética , Células Estreladas do Fígado/metabolismo , Humanos , Neoplasias Hepáticas/secundário , Masculino , Proteínas de Membrana , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-cbl/genética , RNA não Traduzido , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Canais de Cátion TRPM/genética , Fatores de Transcrição/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Sequenciamento Completo do GenomaRESUMO
Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.
Assuntos
Genoma Viral/fisiologia , Hepacivirus/fisiologia , Lipídeos de Membrana/metabolismo , Proteínas do Core Viral/metabolismo , Vírion/fisiologia , Montagem de Vírus/fisiologia , Linhagem Celular , Humanos , Estrutura Terciária de Proteína/fisiologia , Proteínas do Core Viral/genéticaRESUMO
Chronic liver disease caused by infection with hepatitis C virus (HCV) is an important global health problem that currently affects 170 million people. A major impediment in HCV research and drug development has been the lack of culture systems supporting virus production. This obstacle was recently overcome by using JFH1-based full-length genomes that allow production of viruses infectious both in vitro and in vivo. Although this improvement was important, because of the restriction to the JFH1 isolate and a single chimera consisting of J6CF and JFH1-derived sequences, broadly based comparative studies between different HCV strains were not possible. Therefore, in this study we created a series of further chimeric genomes allowing production of infectious genotype (GT) 1a, 1b, 2a, and 3a particles. With the exception of the GT3a/JFH1 chimera, efficient virus production was obtained when the genome fragments were fused via a site located right after the first transmembrane domain of NS2. The most efficient construct is a GT2a/2a chimera consisting of J6CF- and JFH1-derived sequences connected via this junction. This hybrid, designated Jc1, yielded infectious titers 100- to 1,000-fold higher than the parental isolate and all other chimeras, suggesting that determinants within the structural proteins govern kinetic and efficiency of virus assembly and release. Finally, we describe an E1-specific antiserum capable of neutralizing infectivity of all HCV chimeras.