RESUMO
Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.
Assuntos
Lesão Pulmonar , Camundongos , Animais , Proteínas Wnt , Receptores Frizzled , Via de Sinalização Wnt , Células Epiteliais Alveolares , Células-TroncoRESUMO
The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Processamento Alternativo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Isoformas de Proteínas , Fatores de Processamento de RNA , SpliceossomosRESUMO
KEY MESSAGE: A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Plantas/genética , Solanum lycopersicum/genética , cis-trans-Isomerases/genética , Agrobacterium/genética , Carotenoides/metabolismo , Clorofila/genética , Clorofila/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica de Plantas , Licopeno/metabolismo , Solanum lycopersicum/fisiologia , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , cis-trans-Isomerases/metabolismoRESUMO
BACKGROUND: Data from the 1000 Genomes project is quite often used as a reference for human genomic analysis. However, its accuracy needs to be assessed to understand the quality of predictions made using this reference. We present here an assessment of the genotyping, phasing, and imputation accuracy data in the 1000 Genomes project. We compare the phased haplotype calls from the 1000 Genomes project to experimentally phased haplotypes for 28 of the same individuals sequenced using the 10X Genomics platform. RESULTS: We observe that phasing and imputation for rare variants are unreliable, which likely reflects the limited sample size of the 1000 Genomes project data. Further, it appears that using a population specific reference panel does not improve the accuracy of imputation over using the entire 1000 Genomes data set as a reference panel. We also note that the error rates and trends depend on the choice of definition of error, and hence any error reporting needs to take these definitions into account. CONCLUSIONS: The quality of the 1000 Genomes data needs to be considered while using this database for further studies. This work presents an analysis that can be used for these assessments.
Assuntos
Genoma Humano/genética , Haplótipos/genética , Grupos Raciais/genética , Frequência do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala , Projeto Genoma Humano , Humanos , Polimorfismo de Nucleotídeo Único , Grupos Raciais/etnologia , Erro Científico ExperimentalRESUMO
BACKGROUND: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/metabolismo , Vírus do Tumor Mamário do Camundongo , Mutação , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Infecções Tumorais por Vírus/complicações , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Transformação Celular Viral , Classe I de Fosfatidilinositol 3-Quinases/genética , Modelos Animais de Doenças , Feminino , Marcação de Genes , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteína Supressora de Tumor p53/genética , Infecções Tumorais por Vírus/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença/epidemiologia , Adolescente , Adulto , Estudos de Coortes , Exoma , Feminino , Biblioteca Gênica , Genômica , Hemoglobinas Glicadas/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Índia/epidemiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Análise de Sequência de DNA , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Adulto JovemRESUMO
Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.
Assuntos
Neoplasias do Colo/genética , Fusão Gênica/genética , Genes Neoplásicos/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Trombospondinas/genética , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Variações do Número de Cópias de DNA/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Exoma/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Genes APC , Humanos , Fator de Crescimento Insulin-Like II/genética , Dados de Sequência Molecular , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptor ErbB-3/genética , Análise de Sequência de RNA , Transdução de Sinais/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/metabolismoRESUMO
BACKGROUND: Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. RESULTS: Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. CONCLUSIONS: Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanotecnologia/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Análise Serial de Tecidos/métodos , Linhagem Celular , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismoRESUMO
Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Fibroblastos , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel/farmacologia , Farmacogenética , Fosforilação/efeitos dos fármacos , Poliploidia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Vincristina/farmacologiaRESUMO
Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/antagonistas & inibidores , Quinases raf/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Indenos/farmacologia , Indóis/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/química , Quinases raf/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung carcinomas in smokers being the predominant form of the disease. Although previous studies have identified important common somatic mutations in lung cancers, they have primarily focused on a limited set of genes and have thus provided a constrained view of the mutational spectrum. Recent cancer sequencing efforts have used next-generation sequencing technologies to provide a genome-wide view of mutations in leukaemia, breast cancer and cancer cell lines. Here we present the complete sequences of a primary lung tumour (60x coverage) and adjacent normal tissue (46x). Comparing the two genomes, we identify a wide variety of somatic variations, including >50,000 high-confidence single nucleotide variants. We validated 530 somatic single nucleotide variants in this tumour, including one in the KRAS proto-oncogene and 391 others in coding regions, as well as 43 large-scale structural variations. These constitute a large set of new somatic mutations and yield an estimated 17.7 per megabase genome-wide somatic mutation rate. Notably, we observe a distinct pattern of selection against mutations within expressed genes compared to non-expressed genes and in promoter regions up to 5 kilobases upstream of all protein-coding genes. Furthermore, we observe a higher rate of amino acid-changing mutations in kinase genes. We present a comprehensive view of somatic alterations in a single lung tumour, and provide the first evidence, to our knowledge, of distinct selective pressures present within the tumour environment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genoma Humano/genética , Neoplasias Pulmonares/genética , Mutação Puntual/genética , Análise Mutacional de DNA , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proto-Oncogene Mas , Seleção Genética/genéticaRESUMO
The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.
Assuntos
Genes Neoplásicos/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , MAP Quinase Quinase 4/genética , Masculino , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/genética , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas Quinases/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
Hepatitis B virus (HBV) infection is a leading risk factor for hepatocellular carcinoma (HCC). HBV integration into the host genome has been reported, but its scale, impact and contribution to HCC development is not clear. Here, we sequenced the tumor and nontumor genomes (>80× coverage) and transcriptomes of four HCC patients and identified 255 HBV integration sites. Increased sequencing to 240× coverage revealed a proportionally higher number of integration sites. Clonal expansion of HBV-integrated hepatocytes was found specifically in tumor samples. We observe a diverse collection of genomic perturbations near viral integration sites, including direct gene disruption, viral promoter-driven human transcription, viral-human transcript fusion, and DNA copy number alteration. Thus, we report the most comprehensive characterization of HBV integration in hepatocellular carcinoma patients. Such widespread random viral integration will likely increase carcinogenic opportunities in HBV-infected individuals.
Assuntos
Carcinoma Hepatocelular/genética , Genoma Humano/genética , Vírus da Hepatite B/genética , Hepatite B/genética , Neoplasias Hepáticas/genética , Integração Viral/genética , Sequência de Bases , Sítios de Ligação/genética , Carcinoma Hepatocelular/virologia , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Neoplasias Hepáticas/virologia , Masculino , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA/métodos , Transcriptoma/genéticaRESUMO
Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.
Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Neoplasias Pulmonares/genética , Mutação , Transcriptoma , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Éxons , Marcadores Genéticos , Heterozigoto , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Cariotipagem/métodos , Neoplasias Pulmonares/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.
Assuntos
Neoplasias/enzimologia , Neoplasias/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Sequência Conservada , Endocitose/genética , Evolução Molecular , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.
Assuntos
Apoptose/genética , Cromossomos Humanos Par 8/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Humano/genética , Família Multigênica/genética , Neoplasias/genética , Receptores de Morte Celular/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Western Blotting , Linhagem Celular Tumoral , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Testes Genéticos , Humanos , Análise em Microsséries , Neoplasias/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Morte Celular/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: With over 1.3 billion people, India is estimated to contain three times more genetic diversity than does Europe. Next-generation sequencing technologies have facilitated the understanding of diversity by enabling whole genome sequencing at greater speed and lower cost. While genomes from people of European and Asian descent have been sequenced, only recently has a single male genome from the Indian subcontinent been published at sufficient depth and coverage. In this study we have sequenced and analyzed the genome of a South Asian Indian female (SAIF) from the Indian state of Kerala. RESULTS: We identified over 3.4 million SNPs in this genome including over 89,873 private variations. Comparison of the SAIF genome with several published personal genomes revealed that this individual shared ~50% of the SNPs with each of these genomes. Analysis of the SAIF mitochondrial genome showed that it was closely related to the U1 haplogroup which has been previously observed in Kerala. We assessed the SAIF genome for SNPs with health and disease consequences and found that the individual was at a higher risk for multiple sclerosis and a few other diseases. In analyzing SNPs that modulate drug response, we found a variation that predicts a favorable response to metformin, a drug used to treat diabetes. SNPs predictive of adverse reaction to warfarin indicated that the SAIF individual is not at risk for bleeding if treated with typical doses of warfarin. In addition, we report the presence of several additional SNPs of medical relevance. CONCLUSIONS: This is the first study to report the complete whole genome sequence of a female from the state of Kerala in India. The availability of this complete genome and variants will further aid studies aimed at understanding genetic diversity, identifying clinically relevant changes and assessing disease burden in the Indian population.
Assuntos
Povo Asiático/genética , Mapeamento Cromossômico , Genoma Humano , Genoma Mitocondrial , Polimorfismo de Nucleotídeo Único , Anticoagulantes/efeitos adversos , Variações do Número de Cópias de DNA , Diabetes Mellitus/genética , Diabetes Mellitus/prevenção & controle , Feminino , Predisposição Genética para Doença , Variação Genética , Haplótipos , Hemorragia/induzido quimicamente , Hemorragia/genética , Hemorragia/prevenção & controle , Humanos , Hipoglicemiantes/uso terapêutico , Índia , Metformina/uso terapêutico , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/prevenção & controle , Análise de Sequência de DNA , Varfarina/efeitos adversosRESUMO
SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.