RESUMO
Antiviral responses are barriers that must be overcome for efficacy of oncolytic virotherapy. In mammalian cells, antiviral responses involve the interferon pathway, a protein-signaling cascade that alerts the immune system and limits virus propagation. Tumour-specific defects in interferon signaling enhance viral infection and responses to oncolytic virotherapy, but many human cancers are still refractory to oncolytic viruses. Given that invertebrates, fungi and plants rely on RNA interference pathways for antiviral protection, we investigated the potential involvement of this alternative antiviral mechanism in cancer cells. Here, we detected viral genome-derived small RNAs, indicative of RNAi-mediated antiviral responses, in human cancer cells. As viruses may encode suppressors of the RNA interference pathways, we engineered an oncolytic vesicular stomatitis virus variant to encode the Nodamura virus protein B2, a known inhibitor of RNAi-mediated immune responses. B2-expressing oncolytic virus showed enhanced viral replication and cytotoxicity, impaired viral genome cleavage and altered microRNA processing in cancer cells. Our data establish the improved therapeutic potential of our novel virus which targets the RNAi-mediated antiviral defense of cancer cells.
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Vetores Genéticos , Neoplasias/genética , Nodaviridae , Terapia Viral Oncolítica , Vírus Oncolíticos , Interferência de RNA , Animais , Citocinas/metabolismo , Vetores Genéticos/genética , Genoma Viral , Humanos , Interferon Tipo I/metabolismo , Neoplasias/terapia , Nodaviridae/genética , Nodaviridae/metabolismo , Vírus Oncolíticos/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Genes involved in fetal lung development are thought to play crucial roles in the malignant transformation of adult lung cells. Consequently, the study of lung tumour biology in the context of lung development has the potential to reveal key developmentally relevant genes that play critical roles in lung cancer initiation/progression. Here, we describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, with subsequent identification of 37 miRNAs in non-small cell lung cancer (NSCLC) that recapitulate their fetal expression patterns. Nuclear factor I/B (NFIB), a transcription factor essential for lung development, was identified as a potential frequent target for these 'oncofetal' miRNAs. Concordantly, analysis of NFIB expression in multiple NSCLC independent cohorts revealed its recurrent underexpression (in â¼40-70% of tumours). Interrogation of NFIB copy number, methylation, and mutation status revealed that DNA level disruption of this gene is rare, and further supports the notion that oncofetal miRNAs are likely the primary mechanism responsible for NFIB underexpression in NSCLC. Reflecting its functional role in regulating lung differentiation, low expression of NFIB was significantly associated with biologically more aggressive subtypes and, ultimately, poorer survival in lung adenocarcinoma patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Fatores de Transcrição NFI/genética , Invasividade Neoplásica/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Fatores de Transcrição NFI/metabolismo , Invasividade Neoplásica/patologia , Prognóstico , Taxa de SobrevidaRESUMO
Lung cancer is the leading cause of cancer mortality worldwide, and at only 18%, it has one of the lowest 5-year survival rates of all malignancies. With its highly complex mutational landscape, treatment strategies against lung cancer have proved largely ineffective. However with the recent success of immunotherapy trials in lung cancer, there is renewed enthusiasm in targeting the immune component of tumors. Macrophages make up the majority of the immune infiltrate in tumors and are a key cell type linking inflammation and cancer. Although the mechanisms through which inflammation promotes cancer are not fully understood, two connected hypotheses have emerged: an intrinsic pathway, driven by genetic alterations that lead to neoplasia and inflammation, and an extrinsic pathway, driven by inflammatory conditions that increase cancer risk. Here, we discuss the contribution of macrophages to these pathways and subsequently their roles in established tumors. We highlight studies investigating the association of macrophages with lung cancer prognosis and discuss emerging therapeutic strategies for targeting macrophages in the tumor microenvironment.
Assuntos
Imunoterapia/métodos , Inflamação/etiologia , Neoplasias Pulmonares/terapia , Macrófagos/imunologia , Biomarcadores Tumorais/análise , Ensaios Clínicos como Assunto , Progressão da Doença , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Fenótipo , Prognóstico , Taxa de SobrevidaRESUMO
Human PIWI-interacting RNAs (piRNAs) are known to be expressed in germline cells, functionally silencing LINEs and SINEs. Their expression patterns in somatic tissues are largely uncharted. We analyzed 6,260 human piRNA transcriptomes derived from non-malignant and tumour tissues from 11 organs. We discovered that only 273 of the 20,831 known piRNAs are expressed in somatic non-malignant tissues. However, expression patterns of these piRNAs were able to distinguish tissue-of-origin. A total of 522 piRNAs are expressed in corresponding tumour tissues, largely distinguishing tumour from non-malignant tissues in a cancer-type specific manner. Most expressed piRNAs mapped to known transcripts, contrary to "piRNA clusters" reported in germline cells. We showed that piRNA expression can delineate clinical features, such as histological subgroups, disease stages, and survival. PiRNAs common to many cancer types might represent a core gene-set that facilitates cancer growth, while piRNAs unique to individual cancer types likely contribute to cancer-specific biology.
Assuntos
Neoplasias/genética , RNA Interferente Pequeno/metabolismo , Análise por Conglomerados , Genoma Humano , Células Germinativas/metabolismo , Humanos , Estadiamento de Neoplasias , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Análise de Sobrevida , TranscriptomaRESUMO
Microtubule affinity-regulating kinases (MARKs) are involved in several cellular functions but few studies have correlated MARK kinase expression with cancer, and none have explored their role in lung cancer. In this study, we identified MARK2 as frequently disrupted by DNA hypomethylation and copy gain, resulting in concordant overexpression in independent lung tumor cohorts and we demonstrate a role for MARK2 in lung tumor biology. Manipulation of MARK2 in lung cell lines revealed its involvement in cell viability and anchorage-independent growth. Analyses of both manipulated cell lines and clinical tumor specimens identified a potential role for MARK2 in cell cycle activation and DNA repair. Associations between MARK2 and the E2F, Myc/Max and NF-κB pathways were identified by luciferase assays and in-depth assessment of the NF-κB pathway suggests a negative association between MARK2 expression and NF-κB due to activation of non-canonical NF-κB signaling. Finally, we show that high MARK2 expression levels correlate with resistance to cisplatin, a standard first line chemotherapy for lung cancer. Collectively, our work supports a role for MARK2 in promoting malignant phenotypes of lung cancer and potentially modulating response to the DNA damaging chemotherapeutic, cisplatin.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Humanos , Neoplasias Pulmonares/tratamento farmacológico , NF-kappa B/metabolismoRESUMO
Tumor cells harbor tens to thousands of genetic and epigenetic alterations that disrupt cellular pathways, providing them with growth and survival advantages. However, these benefits come at a cost, with uncontrolled cell growth, defective apoptosis, sustained pathological angiogenesis, immune evasion, and a metastatic phenotype occurring at the expense of the antiviral response of the individual tumor cell. Oncolytic virotherapy is an emerging therapeutic strategy that uses replication-competent viruses to selectivity kill cancer cells by exploiting their impaired antiviral response. In this review, we outline our understanding of the alterations in signaling pathways that simultaneously contribute to the malignant phenotype and virus-mediated killing of cancer cells.
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BACKGROUND: The NRF2 pathway has multiple pro-tumorigenic functions, and Nrf2 levels are increased in head and neck squamous cell carcinoma (HNSCC). The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a negative regulator of NRF2. In this study, we investigated mechanisms of disruption of individual complex components. METHODS: Clinical and genomic profiles for 302 patients with HNSCC were obtained from The Cancer Genome Atlas. Combined pattern of epi/genetic alterations for individual components revealed frequent of complex disruption. Gene-set enrichment analysis was performed on expression data to identify affected pathways. RESULTS: DNA loss is the main mechanism of alteration for all component genes, whereas hypermethylation largely affects only KEAP1. Combined analysis revealed that 64% of patients with HNSCC have disruption in this protein complex. Concordantly, NRF2-associated gene signature is enriched in HNSCC. Survival was significantly diminished among patients with one or more disrupted components. CONCLUSION: The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is frequently disrupted in HNSCC by multiple mechanisms. NRF2-based prognostics will benefit from integrated analysis of component genes.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Complexos Ubiquitina-Proteína Ligase/genética , Carcinoma de Células Escamosas/terapia , Proteínas de Transporte/genética , Proteínas Culina/genética , Bases de Dados Factuais , Feminino , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estimativa de Kaplan-Meier , Proteína 1 Associada a ECH Semelhante a Kelch , Masculino , Fator 2 Relacionado a NF-E2/genética , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Transdução de Sinais , Análise de SobrevidaRESUMO
BACKGROUND: Cigarette smoke is associated with the majority of lung cancers: however, 25% of lung cancer patients are non-smokers, and half of all newly diagnosed lung cancer patients are former smokers. Lung tumors exhibit distinct epidemiological, clinical, pathological, and molecular features depending on smoking status, suggesting divergent mechanisms underlie tumorigenesis in smokers and non-smokers. MicroRNAs (miRNAs) are integral contributors to tumorigenesis and mediate biological responses to smoking. Based on the hypothesis that smoking-specific miRNA differences in lung adenocarcinomas reflect distinct tumorigenic processes selected by different smoking and non-smoking environments, we investigated the contribution of miRNA disruption to lung tumor biology and patient outcome in the context of smoking status. METHODS: We applied a whole transcriptome sequencing based approach to interrogate miRNA levels in 94 patient-matched lung adenocarcinoma and non-malignant lung parenchymal tissue pairs from current, former and never smokers. RESULTS: We discovered novel and distinct smoking status-specific patterns of miRNA and miRNA-mediated gene networks, and identified miRNAs that were prognostically significant in a smoking dependent manner. CONCLUSIONS: We conclude that miRNAs disrupted in a smoking status-dependent manner affect distinct cellular pathways and differentially influence lung cancer patient prognosis in current, former and never smokers. Our findings may represent promising biologically relevant markers for lung cancer prognosis or therapeutic intervention.
Assuntos
Adenocarcinoma/etiologia , Adenocarcinoma/mortalidade , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/mortalidade , MicroRNAs/genética , Fumar , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Idoso , Análise por Conglomerados , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Interferência de RNARESUMO
The NFE2-related factor 2 (NRF2) pathway is critical to initiate responses to oxidative stress; however, constitutive activation occurs in different cancer types, including serous ovarian carcinomas (OVCA). The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a regulator of NRF2 levels. Hence, we investigated the DNA-level mechanisms affecting these genes in OVCA. DNA copy-number loss (CNL), promoter hypermethylation, mRNA expression, and sequence mutation for KEAP1, CUL3, and RBX1 were assessed in a cohort of 568 OVCA from The Cancer Genome Atlas. Almost 90% of cases exhibited loss-of-function alterations in any components of the NRF2 inhibitory complex. CNL is the most prominent mechanism of component disruption, with RBX1 being the most frequently disrupted component. These alterations were associated with reduced mRNA expression of complex components, and NRF2 target gene expression was positively enriched in 90% of samples harboring altered complex components. Disruption occurs through a unique DNA-level alteration pattern in OVCA. We conclude that a remarkably high frequency of DNA and mRNA alterations affects components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms. Together, these results suggest a key role for the KEAP1/CUL3/RBX1 complex and NRF2 pathway deregulation in OVCA.
Assuntos
Proteínas de Transporte/genética , Proteínas Culina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias Ovarianas/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Mutação/genética , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
SOX genes are transcription factors with important roles in embryonic development and carcinogenesis. The SOX family of 20 genes is responsible for regulating lineage and tissue specific gene expression patterns, controlling numerous developmental processes including cell differentiation, sex determination, and organogenesis. As is the case with many genes involved in regulating development, SOX genes are frequently deregulated in cancer. In this perspective we provide a brief overview of how SOX proteins can promote or suppress cancer growth. We also present a pan-cancer analysis of aberrant SOX gene expression and highlight potential molecular mechanisms responsible for their disruption in cancer. Our analyses indicate the prominence of SOX deregulation in different cancer types and reveal potential roles for SOX genes not previously described in cancer. Finally, we summarize our recent identification of SOX15 as a candidate tumor suppressor in pancreatic cancer and propose several research avenues to pursue to further delineate the emerging role of SOX15 in development and carcinogenesis.
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BACKGROUND: Reactive oxygen species contribute to normal thyroid function. The NRF2 oxidative response pathway is frequently and constitutively activated in multiple tumor types, including papillary thyroid carcinoma (PTC). Genetic mechanisms underlying NRF2 pathway activation in PTC are not fully understood. Thus, we aimed to determine whether inactivating patterns of DNA-level alterations affect genes encoding for individual NRF2 inhibitor complex components (CUL3/KEAP1/RBX1) occur in PTC. FINDINGS: Combined patterns of epi/genetic alterations for KEAP1/CUL3/RBX1 E3 ubiquitin-ligase complex components were simultaneously interrogated for a panel of 310 PTC cases and 40 adjacent non-malignant tissues. Data were obtained from The Cancer Genome Atlas project. Enrichment of NRF2 pathway activation was assessed by gene-set enrichment analysis using transcriptome data. Our analyses revealed that PTC sustain a strikingly high frequency (80.6%) of disruption to multiple component genes of the NRF2 inhibitor complex. Hypermethylation is the predominant inactivating mechanism primarily affecting KEAP1 (70.6%) and CUL3 (20%), while copy number loss mostly affects RBX1 (16.8%). Concordantly, NRF2-associated gene expression signatures are positively and significantly enriched in PTC. CONCLUSIONS: The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is almost ubiquitously affected by multiple DNA-level mechanisms and downstream NRF2 pathway targets are activated in PTC. Given the importance of this pathway to normal thyroid function as well as to cancer; targeted inhibition of NRF2 regulators may impact strategies for therapeutic intervention involving this pathway.
Assuntos
Carcinoma/enzimologia , Fator 2 Relacionado a NF-E2/genética , Neoplasias da Glândula Tireoide/enzimologia , Ubiquitina-Proteína Ligases/fisiologia , Carcinoma/genética , Carcinoma Papilar , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/genéticaRESUMO
Genetic analyses of lung cancer have helped found new treatments in this disease. We conducted an integrative analysis of gene expression and copy number in 261 non-small cell lung cancers (NSCLC) relative to matched normal tissues to define novel candidate oncogenes, identifying 12q13-15 and more specifically the YEATS4 gene as amplified and overexpressed in ~20% of the NSCLC cases examined. Overexpression of YEATS4 abrogated senescence in human bronchial epithelial cells. Conversely, RNAi-mediated attenuation of YEATS4 in human lung cancer cells reduced their proliferation and tumor growth, impairing colony formation and inducing cellular senescence. These effects were associated with increased levels of p21WAF1 and p53 and cleavage of PARP, implicating YEATS4 as a negative regulator of the p21-p53 pathway. We also found that YEATS4 expression affected cellular responses to cisplastin, with increased levels associated with resistance and decreased levels with sensitivity. Taken together, our findings reveal YEATS4 as a candidate oncogene amplified in NSCLC, and a novel mechanism contributing to NSCLC pathogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Processos de Crescimento Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Amplificação de Genes , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Hibridização de Ácido Nucleico , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
Lung cancer is the leading cause of cancer death worldwide, accounting for more deaths than breast, prostate and colon cancer combined. While treatment decisions are determined primarily by stage, therapeutically non small cell lung cancer (NSCLC) has traditionally been treated as a single disease. However, recent findings have led to the recognition of histology and molecular subtypes as important determinants in treatment selection. Identifying the genetic differences that define these molecular and histological subtypes has the potential to impact treatment and as such is currently the focus of much research. Microarray and genomic sequencing efforts have provided unparalleled insight into the genomes of lung cancer subtypes, specifically adenocarcinoma (AC) and squamous cell carcinoma (SqCC), revealing subtype specific genomic alterations and molecular subtypes as well as differences in cell signaling pathways. In this review, we discuss the recurrent genomic alterations characteristic of AC and SqCC (including molecular subtypes), their therapeutic implications and emerging clinical practices aimed at tailoring treatments based on a tumor's molecular alterations with the hope of improving patient response and survival.
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Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Transdução de SinaisRESUMO
Lung cancer biology has traditionally focused on genomic and epigenomic deregulation of protein-coding genes to identify oncogenes and tumor suppressors diagnostic and therapeutic targets. Another important layer of cancer biology has emerged in the form of noncoding RNAs (ncRNAs), which are major regulators of key cellular processes such as proliferation, RNA splicing, gene regulation, and apoptosis. In the past decade, microRNAs (miRNAs) have moved to the forefront of ncRNA cancer research, while the role of long noncoding RNAs (lncRNAs) is emerging. Here we review the mechanisms by which miRNAs and lncRNAs are deregulated in lung cancer, the technologies that can be applied to detect such alterations, and the clinical potential of these RNA species. An improved comprehension of lung cancer biology will come through the understanding of the interplay between deregulation of non-coding RNAs, the protein-coding genes they regulate, and how these interactions influence cellular networks and signalling pathways.
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For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.
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Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/classificação , Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/classificação , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/classificação , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Progressão da Doença , Epigênese Genética , Genoma , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/patologia , TranscriptomaRESUMO
PURPOSE: Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett's esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers. EXPERIMENTAL DESIGN: Whole genome tiling-path array comparative genomic hybridization was used to identify significant regions of copy number alteration in 20 EACs and 10 matching BE tissues. Copy number and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes. RESULTS: We identified RFC3 as a novel, candidate oncogene activated by amplification in approximately 25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification and was differentially expressed between nonmalignant and EAC tissues. Copy number gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased copy number but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types. CONCLUSIONS: RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker.
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Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Predisposição Genética para Doença/genética , Oncogenes/genética , Proteína de Replicação C/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Estudos de Coortes , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Neoplasias Esofágicas/patologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica/estatística & dados numéricos , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Modelos de Riscos Proporcionais , Interferência de RNARESUMO
INTRODUCTION: Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-ß/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-κB pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer. METHODS: A total of 644 tumor- and 90 cell-line genomes were analyzed for gene dosage status of the individual complex components and IKBKB. Gene expression of these genes and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition. RESULTS: We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. Although both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity. CONCLUSIONS: Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Herein, we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in non-small cell lung cancer.
Assuntos
Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Proteínas Culina/genética , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , NF-kappa B/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/metabolismo , Dosagem de Genes , Perfilação da Expressão Gênica , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Chemotherapy resistance is a key contributor to the dismal prognoses for lung cancer patients. While the majority of studies have focused on sequence mutations and expression changes in protein-coding genes, recent reports have suggested that microRNA (miRNA) expression changes also play an influential role in chemotherapy response. However, the role of genetic alterations at miRNA loci in the context of chemotherapy response has yet to be investigated. In this study, we demonstrate the application of an integrative, multidimensional approach in order to identify miRNAs that are associated with chemotherapeutic resistance and sensitivity utilizing publicly available drug response, miRNA loci copy number, miRNA expression, and mRNA expression data from independent resources. By instigating a logical stepwise strategy, we have identified specific miRNAs that are associated with resistance to several chemotherapeutic agents and provide a proof of principle demonstration of how these various databases may be exploited to derive relevant pharmacogenomic results.
Assuntos
Dosagem de Genes/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MicroRNAs/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Loci Gênicos/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures (1). To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion (2). Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA (3). DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20°C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization (4) (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.
Assuntos
DNA/isolamento & purificação , Inclusão em Parafina/métodos , Extratos de Tecidos/químicaRESUMO
BACKGROUND: An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer) may not be suitable in another (e.g. breast cancer). Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate. METHODS: Serial analysis of gene expression (SAGE) profiles of non-malignant and malignant lung samples were compared using a permutation test to identify the most stably expressed genes across all samples. Subsequently, the specificity of the reference genes was evaluated across multiple tissue types, their constancy of expression was assessed using quantitative RT-PCR (qPCR), and their impact on differential expression analysis of microarray data was evaluated. RESULTS: We show that (i) conventional references genes such as ACTB and GAPDH are highly variable between cancerous and non-cancerous samples, (ii) reference genes identified for lung cancer do not perform well for other cancer types (breast and brain), (iii) reference genes identified through SAGE show low variability using qPCR in a different cohort of samples, and (iv) normalization of a lung cancer gene expression microarray dataset with or without our reference genes, yields different results for differential gene expression and subsequent analyses. Specifically, key established pathways in lung cancer exhibit higher statistical significance using a dataset normalized with our reference genes relative to normalization without using our reference genes. CONCLUSIONS: Our analyses found NDUFA1, RPL19, RAB5C, and RPS18 to occupy the top ranking positions among 15 suitable reference genes optimal for normalization of lung tissue expression data. Significantly, the approach used in this study can be applied to data generated using new generation sequencing platforms for the identification of reference genes optimal within diverse contexts.