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Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking-in part due to the difficulty in assessing migration using high-throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the 'identification of Migration control genes by differential Expression'. MigExpress exploits the combination of in-depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration-associated genes by their differential expression in slow- versus fast-migrating cells. We applied MigExpress to investigate non-small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Proteómica/métodosRESUMEN
The class of circular RNA (circRNA) is characterized by head-to-tail bonds between exons formed by backsplicing. Here, we provide a resource of circRNA expression in a comprehensive panel of 60 lung cancer and non-transformed cell lines (FL3C dataset). RNA sequencing after depletion of ribosomal RNA quantified the expression of circRNA and linear RNA. We detected 148,811 circular RNAs quantified by 2.8 million backsplicing reads originating from 12,251 genes. The number of identified circRNAs was markedly higher using rRNA depletion compared to public polyA-enriched RNA-seq datasets. CircRNAs almost never started in the first exon nor ended in the last exon and started more frequently in earlier exons. Most circRNAs showed high cell line specificity and correlated positively with their linear RNA counterpart. Known cancer genes produced more circRNAs than non-cancer genes. Subsets of circRNAs correlated with cell proliferation, histological subtype or genotype. CircTNFRSF21 was translated crossing the backsplice site in two different reading frames. Overexpression of circPVT1, circERBB2, circHIPK3, circCCNB1, circSMAD2, circTNFRSF21 and circKIF5B significantly increased colony formation. In conclusion, our data provide a comprehensive map of circRNA expression in lung cancer cells and global patterns of circRNA production as a useful resource for future research into lung cancer circRNAs.
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Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.
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Autofagosomas/metabolismo , Virus de la Influenza A/metabolismo , Proteínas Ribosómicas/metabolismo , Autofagia , Línea Celular Tumoral , Humanos , Gripe Humana/metabolismo , Gripe Humana/patología , Gripe Humana/virología , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Ribosomas/metabolismoRESUMEN
Lung cancer continues to be the leading cause of cancer-related deaths worldwide, with little improvement in patient survival rates in the past decade. Long non-coding RNAs (lncRNAs) are gaining importance as possible biomarkers with prognostic potential. By large-scale data mining, we identified LINC00261 as a lncRNA which was significantly downregulated in lung cancer. Low expression of LINC00261 was associated with recurrence and poor patient survival in lung adenocarcinoma. Moreover, the gene pair of LINC00261 and its neighbor FOXA2 were significantly co-regulated. LINC00261 as well as FOXA2 negatively correlated with markers for epithelial-to-mesenchymal transition (EMT) and were suppressed by the EMT inducer TGFß. Hierarchical clustering of gene expression data from lung cancer cell lines could further verify the association of high LINC00261/FOXA2 expression to an epithelial gene signature. Furthermore, higher expression of the LINC00261/FOXA2 locus was associated with lung cancer cell lines with lower migratory capacity. All these data establish LINC00261 and FOXA2 as an epithelial-specific marker pair, downregulated during EMT and lung cancer progression, and associated with lower cell migration potential in lung cancer cells.
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Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers.
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Antígenos de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Animales , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Metástasis de la Neoplasia , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas p21(ras)/genética , Células Tumorales CultivadasRESUMEN
Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.
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Aminoácidos/deficiencia , Autofagia , Biosíntesis de Proteínas , Proteolisis , Autofagosomas/metabolismo , Humanos , Marcaje Isotópico , Células MCF-7RESUMEN
The macroautophagy machinery has been implicated in MHC class II restricted antigen presentation. Here, we report that this machinery assists in the internalization of MHC class I molecules. In the absence of the autophagy factors Atg5 and Atg7, MHC class I surface levels are elevated due to decreased endocytosis and degradation. Internalization of MHC class I molecules occurs less efficiently if AAK1 cannot be recruited via Atg8/LC3B. In the absence of Atg-dependent MHC class I internalization, dendritic cells stimulate CD8(+) T cell responses more efficiently in vitro and in vivo. During viral infections, lack of Atg5 results in enhanced influenza- and LCMV-specific CD8(+) T cell responses in vivo. Elevated influenza-specific CD8(+) T cell responses are associated with better immune control of this infection. Thus, the macroautophagy machinery orchestrates T cell immunity by supporting MHC class II but compromises MHC class I restricted antigen presentation.
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Proteína 5 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/genética , Autofagia/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Presentación de Antígeno/inmunología , Células Cultivadas , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Autophagy is one of the two major degradation pathways within eukaryotic cells. Nevertheless, little is known about the protein composition of autophagosomes, the vesicles shuttling proteins to lysosomes for degradation. Protein correlation profiling in combination with stable isotope labeling by amino acids in cell culture is a stringent method to investigate the dynamics of the autophagosomal proteome. It enables the discrimination between autophagosomal and co-purifying proteins identifying organellar candidate proteins for further investigation.
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Aminoácidos/química , Marcaje Isotópico/métodos , Fagosomas/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteómica/métodos , Métodos Analíticos de la Preparación de la Muestra , Fraccionamiento Celular , Células Cultivadas , Cromatografía Liquida , Espectrometría de Masas , Transporte de ProteínasRESUMEN
Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux. To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction.
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Autofagia/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Línea Celular Tumoral , Humanos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteómica , Inanición/metabolismo , Factores de TiempoRESUMEN
Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Presentación de Antígeno , Autofagia/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas de Microfilamentos/metabolismo , Fagosomas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , NADPH Oxidasas/fisiología , Fagosomas/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A hallmark of macroautophagy is the formation of autophagosomes, double-membrane vesicles that enwrap cellular components destined for lysosomal degradation. We examined autophagosomal protein dynamics under various inducing stimuli using a comprehensive mass spectrometry-based proteomics approach in combination with functional studies in yeast and human cell cultures. Time frame and stimuli type influenced the autophagosome proteome, underlining the dynamic constitution of the organelle. We identified both a core set of proteins always localizing to autophagosomes and stimulus-dependent components that will serve as a resource for further characterization of the autophagosomal machinery and cargo selection. Among the core proteins were newly discovered autophagy regulators found to be conserved from yeast to humans, as well as the proteasome.
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Autofagia , Fagosomas/metabolismo , Proteoma/metabolismo , Señales (Psicología) , Humanos , Modelos Biológicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.
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Autofagia , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Pruebas Genéticas , Fagosomas/metabolismo , Proteínas/metabolismo , Proteómica , Aminoácidos/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antivirales/farmacología , Neoplasias de la Mama/patología , Electroforesis en Gel de Poliacrilamida , Femenino , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Inmunosupresores/farmacología , Marcaje Isotópico , Lisosomas/metabolismo , Macrólidos/farmacología , Fagosomas/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sirolimus/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Inanición , Células Tumorales CultivadasRESUMEN
SIGNIFICANCE: Protein degradation has been identified as being deregulated in numerous human diseases. Hence, proteins involved in proteasomal as well as lysosomal degradation are regarded as interesting potential drug targets and are thoroughly investigated in clinical studies. RECENT ADVANCES: Technical advances in the field of quantitative mass spectrometry (MS)-based proteomics allow for detailed investigations of protein degradation dynamics and identifications of responsible protein-protein interaction networks enabling a systematic analysis of the degradative inventory of the cell and its underlying molecular mechanisms. CRITICAL ISSUES: In the current review we outline recent technical advances and their limitations in MS-based proteomics and discuss their use for the analysis of protein dynamics involved in degradation processes. FUTURE DIRECTIONS: In the next years the analysis of crosstalk between different posttranslational modifications (PTMs) will be a major focus of MS-based proteomics studies. Increasing evidence highlights the complexity of PTMs with positive and negative feedbacks being discovered. In this regard, the generation of absolute quantitative proteomic data will be essential for theoretical scientists to construct predictive network models that constitute a valuable tool for fast hypothesis testing and for explaining underlying molecular mechanisms.
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Proteómica , Espectrometría de MasasRESUMEN
Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2-associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody-based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.