RESUMO
OBJECTIVE: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications. DESIGN: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice. RESULTS: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs. CONCLUSIONS: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.
Assuntos
Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Gástricas/genética , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Fator de Transcrição GATA4/biossíntese , Fator de Transcrição GATA6/biossíntese , Perfilação da Expressão Gênica/métodos , Inativação Gênica , Predisposição Genética para Doença , Xenoenxertos , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Camundongos Nus , Transplante de Neoplasias , Oncogenes/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Almost all gastric cancers are adenocarcinomas, which have considerable heterogeneity among patients. We sought to identify subtypes of gastric adenocarcinomas with particular biological properties and responses to chemotherapy and targeted agents. METHODS: We compared gene expression patterns among 248 gastric tumors; using a robust method of unsupervised clustering, consensus hierarchical clustering with iterative feature selection, we identified 3 major subtypes. We developed a classifier for these subtypes and validated it in 70 tumors from a different population. We identified distinct genomic and epigenomic properties of the subtypes. We determined drug sensitivities of the subtypes in primary tumors using clinical survival data, and in cell lines through high-throughput drug screening. RESULTS: We identified 3 subtypes of gastric adenocarcinoma: proliferative, metabolic, and mesenchymal. Tumors of the proliferative subtype had high levels of genomic instability, TP53 mutations, and DNA hypomethylation. Cancer cells of the metabolic subtype were more sensitive to 5-fluorouracil than the other subtypes. Furthermore, in 2 independent groups of patients, those with tumors of the metabolic subtype appeared to have greater benefits with 5-fluorouracil treatment. Tumors of the mesenchymal subtype contain cells with features of cancer stem cells, and cell lines of this subtype are particularly sensitive to phosphatidylinositol 3-kinase-AKT-mTOR inhibitors in vitro. CONCLUSIONS: Based on gene expression patterns, we classified gastric cancers into 3 subtypes, and validated these in an independent set of tumors. The subgroups have differences in molecular and genetic features and response to therapy; this information might be used to select specific treatment approaches for patients with gastric cancer.
Assuntos
Adenocarcinoma/classificação , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias Gástricas/classificação , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Idoso , Teorema de Bayes , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Estudos de Associação Genética , Humanos , Pessoa de Meia-Idade , Modelos Estatísticos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Análise de Regressão , Estudos Retrospectivos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Análise de Sobrevida , Serina-Treonina Quinases TOR/antagonistas & inibidores , Resultado do TratamentoRESUMO
MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.
Assuntos
Carcinógenos/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , MicroRNAs/genética , Transdução de Sinais/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Ensaios de Triagem em Larga Escala , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/genéticaRESUMO
OBJECTIVE: Cisplatin is a widely used gastric cancer (GC) chemotherapy; however, genetic factors regulating GC responses to cisplatin remain obscure. Identifying genes regulating cisplatin resistance could aid clinicians in tailoring treatments, by distinguishing cisplatin sensitive patients from those who might benefit from alternative platinum therapies, and highlight novel targeted strategies for overcoming cisplatin resistance. Here integrated epigenomics is applied to identify genes associated with GC cisplatin resistance. DESIGN: 20 GC cell lines were subjected to gene expression profiling, DNA methylation profiling and drug response assays. The molecular data were integrated to identify genes highly expressed and unmethylated specifically in cisplatin-resistant lines. Candidate genes were functionally tested by several in vitro and in vivo assays. Clinical impact of candidate genes was also assessed in a cohort of 197 GC patients. RESULTS: Epigenomic analysis identified bone morphogenetic protein 4 (BMP4) as an epigenetically regulated gene highly expressed in cisplatin-resistant lines. Functional assays confirmed that BMP4 is necessary and sufficient for the expression of several prooncogenic traits, likely mediated through stimulation of the epithelial-mesenchymal transition. In primary tumours, BMP4 promoter methylation levels were inversely correlated with BMP4 expression, and patients with high BMP4-expressing tumours exhibited significantly worse prognosis. Therapeutically, targeted genetic inhibition of BMP4 caused significant sensitisation of GC cells to cisplatin. Notably, BMP4-expressing GCs also did not exhibit cross resistance to oxaliplatin. CONCLUSIONS: BMP4 epigenetic and expression status may represent promising biomarkers for GC cisplatin resistance. Targeting BMP4 may sensitise GC cells to cisplatin. Oxaliplatin, a clinically acceptable cisplatin alternative, may represent a potential therapeutic option for BMP4-positive GCs.
Assuntos
Adenocarcinoma/genética , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 4/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gástricas/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Epigenômica/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Prognóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidadeRESUMO
OBJECTIVE: Gastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programmes contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new prognostic factors and reveal novel therapeutic targets. The authors aimed to produce a comprehensive inventory of gene expression programmes expressed in primary GCs, and to identify those expression programmes significantly associated with patient survival. DESIGN: Using a network-modelling approach, the authors performed a large-scale meta-analysis of GC transcriptome data integrating 940 gastric transcriptomes from multiple independent patient cohorts. The authors analysed a training set of 428 GCs and 163 non-malignant gastric samples, and a validation set of 288 GCs and 61 non-malignant gastric samples. RESULTS: The authors identified 178 gene expression programmes ('modules') expressed in primary GCs, which were associated with distinct biological processes, chromosomal location patterns, cis-regulatory motifs and clinicopathological parameters. Expression of a transforming growth factor ß (TGF-ß) signalling associated 'super-module' of stroma-related genes consistently predicted patient survival in multiple GC validation cohorts. The proportion of intra-tumoural stroma, quantified by morphometry in tissue sections from gastrectomy specimens, was also significantly associated with stromal super-module expression and GC patient survival. CONCLUSION: Stromal gene expression predicts GC patient survival in multiple independent cohorts, and may be closely related to the intra-tumoural stroma proportion, a specific morphological GC phenotype. These findings suggest that therapeutic approaches targeting the GC stroma may merit evaluation.
Assuntos
Adenocarcinoma/genética , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Fatores Etários , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/genética , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Genômica/métodos , Humanos , Estadiamento de Neoplasias , Prognóstico , Fatores Sexuais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Estromais/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. DESIGN: Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. RESULTS: 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. CONCLUSION: The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.
Assuntos
Amplificação de Genes , Deleção de Genes , Polimorfismo de Nucleotídeo Único , Neoplasias Gástricas/genética , Antineoplásicos/uso terapêutico , Receptores ErbB/genética , Marcadores Genéticos , Terapia de Alvo Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptor ErbB-2/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/mortalidade , Proteínas ras/genéticaRESUMO
BACKGROUND & AIMS: Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences in patient survival times and responses to various standard-of-care cytotoxic drugs. METHODS: We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-fluorouracil, cisplatin, oxaliplatin) was also assessed. RESULTS: Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF) that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Lauren's histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-fluorouracil and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-fluorouracil-based therapy. CONCLUSIONS: Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Galectina 4/metabolismo , Perfilação da Expressão Gênica , Neoplasias Gástricas/classificação , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Cisplatino/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Prognóstico , Modelos de Riscos Proporcionais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Adulto JovemRESUMO
Many solid cancers are known to exhibit a high degree of heterogeneity in their deregulation of different oncogenic pathways. We sought to identify major oncogenic pathways in gastric cancer (GC) with significant relationships to patient survival. Using gene expression signatures, we devised an in silico strategy to map patterns of oncogenic pathway activation in 301 primary gastric cancers, the second highest cause of global cancer mortality. We identified three oncogenic pathways (proliferation/stem cell, NF-kappaB, and Wnt/beta-catenin) deregulated in the majority (>70%) of gastric cancers. We functionally validated these pathway predictions in a panel of gastric cancer cell lines. Patient stratification by oncogenic pathway combinations showed reproducible and significant survival differences in multiple cohorts, suggesting that pathway interactions may play an important role in influencing disease behavior. Individual GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Predicting pathway activity by expression signatures thus permits the study of multiple cancer-related pathways interacting simultaneously in primary cancers, at a scale not currently achievable by other platforms.
Assuntos
Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Gástricas/metabolismo , Adulto JovemRESUMO
Successful tumor development and progression involves the complex interplay of both pro- and anti-oncogenic signaling pathways. Genetic components balancing these opposing activities are likely to require tight regulation, because even subtle alterations in their expression may disrupt this balance with major consequences for various cancer-associated phenotypes. Here, we describe a cassette of cancer-specific genes exhibiting precise transcriptional control in solid tumors. Mining a database of tumor gene expression profiles from six different tissues, we identified 48 genes exhibiting highly restricted levels of gene expression variation in tumors (n = 270) compared to nonmalignant tissues (n = 71). Comprising genes linked to multiple cancer-related pathways, the restricted expression of this "Poised Gene Cassette" (PGC) was robustly validated across 11 independent cohorts of approximately 1,300 samples from multiple cancer types. In three separate experimental models, subtle alterations in PGC expression were consistently associated with significant differences in metastatic and invasive potential. We functionally confirmed this association in siRNA knockdown experiments of five PGC genes (p53CSV, MAP3K11, MTCH2, CPSF6, and SKIP), which either directly enhanced the invasive capacities or inhibited the proliferation of AGS cancer cells. In primary tumors, similar subtle alterations in PGC expression were also repeatedly associated with clinical outcome in multiple cohorts. Taken collectively, these findings support the existence of a common set of precisely controlled genes in solid tumors. Since inducing small activity changes in these genes may prove sufficient to potently influence various tumor phenotypes such as metastasis, targeting such precisely regulated genes may represent a promising avenue for novel anti-cancer therapies.
Assuntos
Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica/genética , Metástase Neoplásica/fisiopatologia , Neoplasias/metabolismo , Sobrevida , Animais , Estudos de Coortes , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Biologia Computacional/métodos , Bases de Dados Factuais , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Deregulated gene expression is a hallmark of cancer; however, most studies to date have analyzed short-read RNA sequencing data with inherent limitations. Here, we combine PacBio long-read isoform sequencing (Iso-Seq) and Illumina paired-end short-read RNA sequencing to comprehensively survey the transcriptome of gastric cancer (GC), a leading cause of global cancer mortality. RESULTS: We performed full-length transcriptome analysis across 10 GC cell lines covering four major GC molecular subtypes (chromosomal unstable, Epstein-Barr positive, genome stable and microsatellite unstable). We identify 60,239 non-redundant full-length transcripts, of which > 66% are novel compared to current transcriptome databases. Novel isoforms are more likely to be cell line and subtype specific, expressed at lower levels with larger number of exons, with longer isoform/coding sequence lengths. Most novel isoforms utilize an alternate first exon, and compared to other alternative splicing categories, are expressed at higher levels and exhibit higher variability. Collectively, we observe alternate promoter usage in 25% of detected genes, with the majority (84.2%) of known/novel promoter pairs exhibiting potential changes in their coding sequences. Mapping these alternate promoters to TCGA GC samples, we identify several cancer-associated isoforms, including novel variants of oncogenes. Tumor-specific transcript isoforms tend to alter protein coding sequences to a larger extent than other isoforms. Analysis of outcome data suggests that novel isoforms may impart additional prognostic information. CONCLUSIONS: Our results provide a rich resource of full-length transcriptome data for deeper studies of GC and other gastrointestinal malignancies.
Assuntos
Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Transcriptoma , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Linhagem Celular Tumoral , Éxons , Perfilação da Expressão Gênica , Genoma , Humanos , Fases de Leitura Aberta , Isoformas de Proteínas , Análise de Sequência de RNARESUMO
Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.
Assuntos
Epigenômica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Telomerase/biossíntese , Transativadores/metabolismo , Linhagem Celular Tumoral , Humanos , Mutação , Proteínas de Neoplasias/genética , Elementos de Resposta , Neoplasias Gástricas/genética , Telomerase/genética , Transativadores/genéticaRESUMO
G-CSF can induce functional immune tolerance in man. In this study, purified T cells from G-CSF-mobilized peripheral blood stem cell (PBSC) donors were analysed by gene expression profiling and immunophenotyping. Results suggested a predominantly immune tolerant profile with upregulation of genes related to Th2 and Treg cells, downregulation of genes associated with Th1 cells, cytotoxicity, antigen presentation and graft-versus-host disease (GVHD) and overexpression of negative regulators of Th17 differentiation. Immunophenotyping revealed that during G-CSF exposure donors had reduced levels of T cells with a Th17 phenotype (CD4+IL-17A+CCR6+IL-23R+), more than three times lower compared to normal controls. G-CSF also led to increased levels of CD4+CD25highCD45RO+ Treg cells. Furthermore, mRNA levels of RORgammat, a Th17-specific transcription factor, decreased in T cells isolated from G-CSF-mobilized PBSC harvests. Th17 cells have been implicated in autoimmune diseases and GVHD pathophysiology. Our study is the first to report the effect of G-CSF on the Th17 subpopulation.
Assuntos
Perfilação da Expressão Gênica/métodos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Linfócitos T/metabolismo , Doadores de Sangue , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Análise por Conglomerados , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Interleucina-17/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Chromosomal amplifications of the 11q13 genomic region are frequent in head and neck squamous cell carcinoma (HNSCC). To identify novel 11q13 amplification targets, we integrated high-resolution array-based comparative genomic hybridization and Affymetrix gene-expression profiling of eight HNSCC cell lines. We found that PPFIA1 was the highest upregulated gene in the 11q13 amplicon of HNSCC cell lines when compared with HNSCC lines without 11q13 amplification and confirmed the upregulation of PPFIA1 in primary HNSCCs by real-time PCR. Using siRNA knockdown, we investigated PPFIA1 function in three HNSCC lines using both in vitro invasion assays and wound-healing assays. Surprisingly, we found that cancer cells become more invasive when the PPFIA1 protein levels were reduced, suggesting that PPFIA1 may act as an invasion inhibitor in HNSCC. This unexpected result suggests that the 11q13 amplicon may comprise both positive and negative regulators involved in HNSCC. Our study is the first to evaluate the role of PPFIA1 in head and neck carcinogenesis and suggests a potential link between PPFIA1 activity and cell-extracellular matrix interactions. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 11/genética , Amplificação de Genes , Neoplasias de Cabeça e Pescoço/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologiaRESUMO
Purpose: The oxaliplatin plus S-1 and cisplatin plus S-1 regimens are interchangeably used in the management of advanced gastric cancer. The previously reported G-intestinal (G1) and G-diffuse (G2) intrinsic gene expression signatures showed promise for stratifying patients according to their tumor sensitivity to oxaliplatin or cisplatin.Experimental Design: The proof-of-concept, multicenter, open-label phase II "3G" trial was done to prospectively evaluate the feasibility and efficacy of using genomic classifiers to tailor treatment in gastric cancer. Patients' tumors were classified as "G1" or "G2" using a nearest-prediction template method, or "G3" (unclear assignment) when FDR ≥ 0.05. The first 30 patients in the "G1" cohort were assigned oxaliplatin plus S-1 (SOX) chemotherapy; thereafter, subsequently recruited "G1" patients were treated with cisplatin plus S-1 (SP) chemotherapy. "G2" patients and "G3" patients were treated with SP and SOX chemotherapy, respectively.Results: A total of 48, 21, and 12 patients, respectively, were given "G1," "G2," and "G3" genomic assignments. Median turnaround time was 7 days (IQR, 5-9). Response rates were 44.8%, 8.3%, 26.7%, and 55.6% for the "G1-SOX," "G1-SP," "G2," "G3" cohorts, respectively; and was higher in G1 patients treated with SOX compared with SP (P = 0.033). Exploratory analyses using the genomic classifier of Lei and colleagues validated the utility of the metabolic signature as a biomarker for predicting benefit from chemotherapy (log-rank P = 0.004 for PFS), whereas the Asian Cancer Research Group classifier did not demonstrate any predictive value.Conclusions: This bench-to-bedside effort establishes a reasonable turnaround time for gene expression profiling and possible utility of genomic classifiers in gastric cancer treatment stratification. Clin Cancer Res; 24(21); 5272-81. ©2018 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Perfilação da Expressão Gênica , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Transcriptoma , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Feminino , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Gástricas/diagnóstico , Resultado do TratamentoRESUMO
In this paper, we describe the development of a device for fuming fingerprints with cyanoacrylate (Super Glue) to enable police tactical units to obtain fingerprint evidence from suspicious packages using a remote-controlled robot. Through a series of initial experiments and preliminary designs, we show that effective cyanoacylate fuming requires sufficient heat, humidity, and airflow. This work led to the development of a final working prototype, called robot accessory for fuming fingerprint evidence (RAFFE), which is currently being field tested by the Calgary Police Service.
Assuntos
Cianoacrilatos , Dermatoglifia , Medicina Legal/instrumentação , Robótica , Desenho de Equipamento , Medicina Legal/métodos , Humanos , Temperatura , Termodinâmica , VolatilizaçãoRESUMO
Chromatin alterations are fundamental hallmarks of cancer. To study chromatin alterations in primary gastric adenocarcinomas, we perform nanoscale chromatin immunoprecipitation sequencing of multiple histone modifications in five gastric cancers and matched normal tissues. We identify hundreds of somatically altered promoters and predicted enhancers. Many cancer-associated promoters localize to genomic sites lacking previously annotated transcription start sites (cryptic promoters), driving expression of nearby genes involved in gastrointestinal cancer, embryonic development and tissue specification. Cancer-associated promoters overlap with embryonic stem cell regions targeted by polycomb repressive complex 2, exhibiting promoter bivalency and DNA methylation loss. We identify somatically acquired elements exhibiting germline allelic biases and non-coding somatic mutations creating new promoters. Our findings demonstrate the feasibility of profiling chromatin from solid tumours with limited tissue to identify regulatory elements, transcriptional patterns and regulatory genetic variants associated with cancer.
Assuntos
Adenocarcinoma/genética , Cromatina/genética , Impressões Digitais de DNA/métodos , Nanotecnologia/métodos , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética , Neoplasias Gástricas/genética , Adenocarcinoma/patologia , Alelos , Estudos de Casos e Controles , DNA/genética , Metilação de DNA , DNA de Neoplasias/genética , Histonas/genética , Humanos , Mutação/genética , Neoplasias Gástricas/patologia , Sítio de Iniciação de TranscriçãoRESUMO
Epigenetic alterations are fundamental hallmarks of cancer genomes. We surveyed the landscape of DNA methylation alterations in gastric cancer by analyzing genome-wide CG dinucleotide (CpG) methylation profiles of 240 gastric cancers (203 tumors and 37 cell lines) and 94 matched normal gastric tissues. Cancer-specific epigenetic alterations were observed in 44% of CpGs, comprising both tumor hyper- and hypomethylation. Twenty-five percent of the methylation alterations were significantly associated with changes in tumor gene expression. Whereas most methylation-expression correlations were negative, several positively correlated methylation-expression interactions were also observed, associated with CpG sites exhibiting atypical transcription start site distances and gene body localization. Methylation clustering of the tumors revealed a CpG island methylator phenotype (CIMP) subgroup associated with widespread hypermethylation, young patient age, and adverse patient outcome in a disease stage-independent manner. CIMP cell lines displayed sensitivity to 5-aza-2'-deoxycytidine, a clinically approved demethylating drug. We also identified long-range regions of epigenetic silencing (LRESs) in CIMP tumors. Combined analysis of the methylation, gene expression, and drug treatment data suggests that certain LRESs may silence specific genes within the region, rather than all genes. Finally, we discovered regions of long-range tumor hypomethylation, associated with increased chromosomal instability. Our results provide insights into the epigenetic impact of environmental and biological agents on gastric epithelial cells, which may contribute to cancer.
Assuntos
Metilação de DNA , Epigênese Genética , Neoplasias Gástricas/genética , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proliferação de Células , Instabilidade Cromossômica , Análise por Conglomerados , Ilhas de CpG , Decitabina , Epigenômica , Mucosa Gástrica/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Transplante de Neoplasias , FenótipoRESUMO
Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.
Assuntos
Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/parasitologia , Colangiocarcinoma/genética , Colangiocarcinoma/parasitologia , Fasciolíase/complicações , Adulto , Idoso , Proteínas de Ligação a DNA/genética , Exoma , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Mutação , Receptores Imunológicos/genética , Análise de Sequência de DNARESUMO
Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.
Assuntos
Adenocarcinoma/genética , Adesão Celular/genética , Montagem e Desmontagem da Cromatina/genética , Exoma/genética , Genes Supressores de Tumor , Mutação/genética , Neoplasias Gástricas/genética , Estudos de Casos e Controles , DNA/genética , Mucosa Gástrica/metabolismo , Humanos , Instabilidade de Microssatélites , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNARESUMO
PURPOSE: East-Asian (EA) patients with non-small-cell lung cancer (NSCLC) are associated with a high proportion of nonsmoking women, epidermal growth factor receptor (EGFR)-activating somatic mutations, and clinical responses to tyrosine kinase inhibitors. We sought to identify novel molecular differences between NSCLCs from EA and Western European (WE) patients. EXPERIMENTAL DESIGN: A total of 226 lung adenocarcinoma samples from EA (n = 90) and WE (n = 136) patients were analyzed for copy number aberrations (CNA) by using a common high-resolution SNP (single nucleotide polymorphism) microarray platform. Univariate and multivariate analyses were carried out to identify CNAs specifically related to smoking history, EGFR mutation status, and ethnicity. RESULTS: The overall genomic profiles of adenocarcinomas from EA and WE patients were highly similar. Univariate analyses revealed several CNAs significantly associated with ethnicity, EGFR mutation, and smoking, but not to gender, and KRAS or p53 mutations. A multivariate model identified four ethnic-specific recurrent CNAs-significantly higher rates of copy number gain were observed on 16p13.13 and 16p13.11 in EA tumors, whereas higher rates of genomic loss on 19p13.3 and 19p13.11 were observed in tumors from WE patients. We identified several potential driver genes in these regions, showing a positive correlation between cis-localized copy number changes and transcriptomic changes. CONCLUSION: 16p copy number gains (EA) and 19p losses (WE) are ethnic-specific chromosomal aberrations in lung adenocarcinoma. Patient ethnicity should be considered when evaluating future NSCLC therapies targeting genes located on these areas.