RESUMEN
BACKGROUND: Upstream open reading frames (uORFs) initiate translation within mRNA 5' leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5' leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in a model of neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. RESULTS: Using a spectral coherence algorithm (SPECtre), we identify 4954 consistently translated uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. On a population basis, the global impact of both AUG and non-AUG initiated uORFs on basal CDS translation were small, even when analysis is limited to conserved and consistently translated uORFs. However, uORFs did alter the translation of a subset of genes, including the Diamond-Blackfan Anemia associated ribosomal gene RPS24. With retinoic acid induced differentiation, we observed an overall positive correlation in translational shifts between uORF/CDS pairs. However, CDSs downstream of uORFs show smaller shifts in TE with differentiation relative to CDSs without a predicted uORF, suggesting that uORF translation buffers cell state dependent fluctuations in CDS translation. CONCLUSION: This work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation.
Asunto(s)
Diferenciación Celular/genética , Neuronas/metabolismo , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Algoritmos , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Neuronas/citología , Ribosomas/metabolismoRESUMEN
BACKGROUND: Active protein translation can be assessed and measured using ribosome profiling sequencing strategies. Prevailing analytical approaches applied to this technology make use of sequence fragment length profiling or reading frame occupancy enrichment to differentiate between active translation and background noise, however they do not consider additional characteristics inherent to the technology which limits their overall accuracy. RESULTS: Here, we present an analytical tool that models the overall tri-nucleotide periodicity of ribosomal occupancy using a classifier based on spectral coherence. Our software, SPECtre, examines the relationship of normalized ribosome profiling read coverage over a rolling series of windows along a transcript relative to an idealized reference signal without the matched requirement of mRNA-Seq. CONCLUSIONS: A comparison of SPECtre against previously published methods on existing data shows a marked improvement in accuracy for detecting active translation and exhibits overall high accuracy at a low false discovery rate. In addition, SPECtre performs comparably to a recently published method similarly based on spectral coherence, however with reduced runtime and memory requirements. SPECtre is available as an open source software package at https://github.com/mills-lab/spectreok .
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Algoritmos , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Transcriptoma/genética , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/genética , Ribosomas/genéticaRESUMEN
Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.
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Neoplasias de la Próstata/genética , Proliferación Celular , Células Cultivadas , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Orquiectomía , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Alineación de Secuencia , Transducción de SeñalRESUMEN
Cancer cells undergo significant metabolic adaptation. Cellular transformation enhances both glycolysis and mitochondrial respiration efficiency through the induction of HIF-1α and HIF-2α. In this process, energy production and synthesis of macromolecules are maximized with minimal ROS accumulation. Furthermore, a series of antioxidant enzymes are induced to mitigate the damaging effects of ROS. Examination of these metabolic changes provides rationale for a synergistic approach to combination anti-cancer therapy; targeted inhibition of HIF and inhibition of cellular defenses against oxidative stress.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/metabolismo , Especies Reactivas de Oxígeno , Antineoplásicos/farmacología , Antioxidantes/metabolismo , Glucosa/metabolismo , Glucólisis , Humanos , Sustancias Macromoleculares , Mitocondrias/metabolismo , Modelos Biológicos , Estrés OxidativoRESUMEN
BACKGROUND: Activating KRAS mutations are important for cancer initiation and progression; and have recently been shown to cause primary resistance to therapies targeting the epidermal growth factor receptor. Therefore, strategies are currently in development to overcome treatment resistance due to oncogenic KRAS. The hypoxia-inducible factors-1α and -2α (HIF-1α and HIF-2α) are activated in cancer due to dysregulated ras signaling. METHODS: To understand the individual and combined roles of HIF-1α and HIF-2α in cancer metabolism and oncogenic KRAS signaling, we used targeted homologous recombination to disrupt the oncogenic KRAS, HIF-1α, and HIF-2α gene loci in HCT116 colon cancer cells to generate isogenic HCT116WT KRAS, HCT116HIF-1α-/-, HCT116HIF-2α-/-, and HCT116HIF-1α-/-HIF-2α-/- cell lines. RESULTS: Global gene expression analyses of these cell lines reveal that HIF-1α and HIF-2α work together to modulate cancer metabolism and regulate genes signature overlapping with oncogenic KRAS. Cancer cells with disruption of both HIF-1α and HIF-2α or oncogenic KRAS showed decreased aerobic respiration and ATP production, with increased ROS generation. CONCLUSION: Our findings suggest novel strategies for treating tumors with oncogenic KRAS mutations.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias del Colon/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Adenosina Trifosfato/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Línea Celular Tumoral , Neoplasias del Colon/genética , Perfilación de la Expresión Génica , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mutagénesis Sitio-Dirigida , Consumo de Oxígeno/genética , Consumo de Oxígeno/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas ras/genéticaRESUMEN
Sunitinib is an oral small-molecule multitargeted receptor tyrosine kinase inhibitor that has recently been shown to have clinical benefit as a single agent in renal cell cancer and gastrointestinal stromal tumors, leading to its Food and Drug Administration approval for treatment of these cancers. However, the benefit is short-lived; and for the majority of cancers, sunitinib single-agent clinical activity is low. Therefore, combination strategies with sunitinib are currently in clinical development. The hypoxia-inducible transcription factors, HIF-1 and HIF-2, induce gene programs important for cancer cell growth and angiogenesis. We hypothesized that inhibiting HIF-1 and HIF-2 would further improve tumor response to sunitinib therapy. To test this hypothesis, HIF-1α and HIF-2α genes were disrupted in colon cancer cells. We found that disruption of HIF-1α, HIF-2α, or both HIF-1α and HIF-2α genes led to improved tumor response to sunitinib. For xenografts in which both HIF-1α and HIF-2α genes were disrupted, there was prolonged complete remission with sunitinib treatment in 50% of mice. This enhanced response was mediated by two potential mechanisms. First, tumor angiogenesis and perfusion were almost completely inhibited by sunitinib when both HIF-1α and HIF-2α genes were disrupted. The enhanced inhibitory effect on tumor angiogenesis was mediated by the inhibition of multiple proangiogenic factors, including vascular endothelial growth factor and angiopoietin-like protein 4, and the induction of the antiangiogenic factor, thrombospondin 1. Second, disruption of HIF-1α, HIF-2α, or both HIF-1α and HIF-2α genes directly inhibited tumor cell proliferation. These preclinical findings have clinical implications and suggest novel clinical trials.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias del Colon/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Indoles/farmacología , Pirroles/farmacología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Indoles/uso terapéutico , Ratones , Ratones Desnudos , Neovascularización Patológica , Pirroles/uso terapéutico , Sunitinib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antiangiogenic therapy improves survival in patients with advanced stage cancers. Currently, there are no reliable predictors or markers for tumor vessel response to antiangiogenic therapy. To model effective antiangiogenic therapy, we disrupted the VEGF gene in three representative cancer cell lines. HCT116 xenografts had low proportions of endothelial tubes covered by pericytes that stained with alpha-smooth muscle actin (SMA) antibody. Upon disruption of VEGF, HCT116(VEGF-/-) xenografts had significantly decreased tumor microvessel perfusion compared with their parental counterparts. Furthermore, HCT116(VEGF-/-) xenografts mounted a tumor-reactive response to hypoxia, characterized by the induction of hypoxia-inducible factor-1 (HIF-1) target genes. One highly induced protein was DPP4, a measurable serum protein that has well-described roles in cancer progression. In contrast, LS174T and MKN45 tumor xenografts had high proportion of endothelial tubes that were covered by SMA+ pericytes. Upon disruption of VEGF, LS174T(VEGF-/-) and MKN45(VEGF-/-) xenografts maintained tumor microvessel perfusion. As such, there were no changes in intratumoral hypoxia or HIF-1 alpha induction. Together, these data show that the extent of tumor vessel response to angiogenic inhibition could be correlated with (a) the preexisting coverage of tumor endothelial tubes with SMA+ pericytes and (b) differential tumor induction of HIF-1 target genes. The data further show that DPP4 is a novel marker of HIF-1 induction. Altogether, these preclinical findings suggest novel clinical trials for predicting and monitoring tumor vessel responses to antiangiogenic therapy.