RESUMEN
Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.
Asunto(s)
Adenocarcinoma , Neoplasias Pancreáticas , Adenocarcinoma/metabolismo , Animales , Matriz Extracelular , Humanos , Hidrogeles/metabolismo , Ratones , Organoides , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Microambiente TumoralRESUMEN
The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.
Asunto(s)
Mitosis , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Proteína Quinasa CDC2/metabolismo , Segregación Cromosómica , Secuencia Conservada , Ciclina B/metabolismo , Activación Enzimática , Células HeLa , Holoenzimas/metabolismo , Humanos , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Fosforilación , Proteína Fosfatasa 2/química , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de SeñalRESUMEN
The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.
Asunto(s)
Membrana Celular/metabolismo , Regulación hacia Abajo , Herpesvirus Humano 8/enzimología , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas con Dominio MARVEL/biosíntesis , Proteolípidos/biosíntesis , Ubiquitina-Proteína Ligasas/biosíntesis , Proteínas Virales/biosíntesis , Membrana Celular/genética , Membrana Celular/inmunología , Pruebas Genéticas , Células HeLa , Células Hep G2 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/inmunología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas con Dominio MARVEL/genética , Proteínas con Dominio MARVEL/inmunología , Proteolípidos/genética , Proteolípidos/inmunología , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/inmunología , Sarcoma de Kaposi/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
RATIONALE: Mapping sites of wild-type SUMO modification is a challenging endeavour. Here we postulate that a combination of chemical derivatistation and collision-induced dissociation (CID) could be used to generate SUMO remnant diagnostic ions to aid both detection of these isopeptides and increase the analytical value of the product ion spectra required to characterize the nature and position of modification. METHODS: SUMO(2/3)ylated proteins were digested with trypsin to generate isopeptides bearing TGG and QTGG isotags. The resulting digests were then dimethyl labelled followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) utilising CID in a data-dependent acquisition on a QSTAR XL. Product ion spectra were interrogated for the presence of iso-N-terminal fragment ions in addition to backbone sequence ions. The ability to diagnostically detect these isopeptides was tested by generation of co-XICs of the iso-N-terminal fragments in a semi-complex background. RESULTS: Dimethyl labelling facilitated the robust detection of a1', b2' & b3' (TGG isotag) and a1', b2' & b4' (QTGG isotag) ions. The abundance of both N-terminal and iso-N-terminal fragment ions, supported by dimethyl labelling, facilitated the generation of information-rich product ion spectra of these isopeptides to aid confident site assignment. Moreover, the diagnostic nature of the combined XICs of the iso-N-terminal fragments supported detection of the isopeptide signals from a semi-complex background. CONCLUSIONS: A combination of dimethyl labelling and CID does indeed lead to the generation of SUMO remnant isopeptide product ion spectra which are more analytically rich. This enables an improvement in characterization of both the isotag and backbone sequences and the site of modification. The diagnostic value of iso-N-terminal fragment ions allows for post-acquisition XIC interrogation to detect putative isopeptides of interest.
Asunto(s)
Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Mapeo Peptídico , Sumoilación , Espectrometría de Masas en Tándem , Tripsina/químicaRESUMEN
RATIONALE: Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. METHODS: SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole-time-of-flight (Q-TOF)-based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO-derived fragments. The utility of this approach was demonstrated with di-SUMO 2, di-SUMO 3, SUMO 1-RanGap(418-587) 1 and an enriched population of SUMOylated proteins. RESULTS: Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di-SUMO-2/3), TGG (di-SUMO-2/3) and GG (SUMO-1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO-modified proteins enriched from a cell lysate. CONCLUSIONS: This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis.
Asunto(s)
Bases de Datos de Proteínas , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Sumoilación , Secuencia de Aminoácidos , Cromatografía Liquida , Biología Computacional , Células HEK293 , Humanos , Lisina/química , Lisina/metabolismo , Datos de Secuencia Molecular , Elastasa Pancreática/metabolismo , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Proteínas/análisis , Proteínas/química , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Tripsina/metabolismoRESUMEN
Congenital disorders of glycosylation (CDG) are rare genetic disorders with a spectrum of clinical manifestations caused by abnormal N-glycosylation of secreted and cell surface proteins. Over 130 genes are implicated and next generation sequencing further identifies potential disease drivers in affected individuals. However, functional testing of these variants is challenging, making it difficult to distinguish pathogenic from non-pathogenic events. Using proximity labelling, we identified OST48 as a protein that transiently interacts with lysyl oxidase (LOX), a secreted enzyme that cross-links the fibrous extracellular matrix. OST48 is a non-catalytic component of the oligosaccharyltransferase (OST) complex, which transfers glycans to substrate proteins. OST48 is encoded by DDOST, and 43 variants of DDOST are described in CDG patients, of which 34 are classified as variants of uncertain clinical significance (VUS). We developed an assay based on LOX N-glycosylation that confirmed two previously characterised DDOST variants as pathogenic. Notably, 39 of the 41 remaining variants did not have impaired activity, but we demonstrated that p.S243F and p.E286del were functionally impaired, consistent with a role in driving CDG in those patients. Thus, we describe a rapid assay for functional testing of clinically relevant CDG variants to complement genome sequencing and support clinical diagnosis of affected individuals.
Asunto(s)
Trastornos Congénitos de Glicosilación , Humanos , Glicosilación , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Relevancia Clínica , Secuencia de Bases , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.
Asunto(s)
Membrana Celular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Receptores de LDL/metabolismo , Animales , Antígenos CD/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Regulación hacia Abajo , Integrinas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo de Interacción de Proteínas , Proteómica , Receptores de LDL/genética , Espectrometría de Masas en Tándem , Receptores Toll-Like/metabolismoRESUMEN
Embryonic stem (ES) cells can differentiate in vitro to produce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of Bry(GFP/+) ES cell to hemangioblasts can be followed by the expression of the Bry(GFP/+) and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating Bry(GFP) ES cells were obtained by flow cytometric sorting, GFP-Flk1- (epiblast), GFP+Flk1- (mesoderm), and GFP+Flk1+ (hemangioblast). Microarray analyses and relative quantification two-dimensional LCLC-MS/MS on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probe set. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4, and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and Western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation, and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Proteómica/métodos , Animales , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Redes y Vías Metabólicas , Ratones , Análisis por Matrices de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Espectrometría de Masas en Tándem , Transcripción Genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Histona Demetilasas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The fission yeast scaffold molecule Sid4 anchors the septum initiation network to the spindle pole body (SPB, centrosome equivalent) to control mitotic exit events. A second SPB-associated scaffold, Cut12, promotes SPB-associated Cdk1-cyclin B to drive mitotic commitment. Signals emanating from each scaffold have been assumed to operate independently to promote two distinct outcomes. We now find that signals from Sid4 contribute to the Cut12 mitotic commitment switch. Specifically, phosphorylation of Sid4 by NIMAFin1 reduces Sid4 affinity for its SPB anchor, Ppc89, while also enhancing Sid4's affinity for casein kinase 1δ (CK1δ). The resulting phosphorylation of Sid4 by the newly docked CK1δ recruits Chk2Cds1 to Sid4. Chk2Cds1 then expels the Cdk1-cyclin B antagonistic phosphatase Flp1/Clp1 from the SPB. Flp1/Clp1 departure can then support mitotic commitment when Cdk1-cyclin B activation at the SPB is compromised by reduction of Cut12 function. Such integration of signals emanating from neighboring scaffolds shows how centrosomes/SPBs can integrate inputs from multiple pathways to control cell fate.
Asunto(s)
Centrosoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cuerpos Polares del Huso/metabolismo , Sitios de Unión , Quinasa Idelta de la Caseína/genética , Quinasa Idelta de la Caseína/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Mutación , Quinasa 1 Relacionada con NIMA/genética , Quinasa 1 Relacionada con NIMA/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal , Cuerpos Polares del Huso/genética , Factores de TiempoRESUMEN
The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection. We applied plasma membrane profiling, a technique that combines quantitative mass spectrometry with selective cell surface aminooxy-biotinylation, to Bacille Calmette-Guérin (BCG)-infected THP-1 macrophages. We quantified 559 PM proteins in BCG-infected THP-1 cells. One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1. We showed that ABCA1 was upregulated on the macrophage cell-surface following infection with pathogenic mycobacteria and knockdown of ABCA1 resulted in increased mycobacterial survival within macrophages, suggesting that it may be a novel mycobacterial host-restriction factor.
RESUMEN
Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.
Asunto(s)
Neoplasias Colorrectales/metabolismo , GTP Fosfohidrolasas/química , Regulación Neoplásica de la Expresión Génica , Genes ras , Proteínas de la Membrana/química , Proteínas Proto-Oncogénicas p21(ras)/química , Aminoácidos/química , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Humanos , Espectrometría de Masas , Mutación , Isoformas de Proteínas/química , Proteómica , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Cancer cells grow in highly complex stromal microenvironments, which through metabolic remodelling, catabolism, autophagy and inflammation nurture them and are able to facilitate metastasis and resistance to therapy. However, these changes in the metabolic profile of stromal cancer-associated fibroblasts and their impact on cancer initiation, progression and metastasis are not well-known. This is the first study to provide a comprehensive proteomic portrait of the azathioprine and taxol-induced catabolic state on human stromal fibroblasts, which comprises changes in the expression of metabolic enzymes, myofibroblastic differentiation markers, antioxidants, proteins involved in autophagy, senescence, vesicle trafficking and protein degradation, and inducers of inflammation. Interestingly, many of these features are major contributors to the aging process. A catabolic stroma signature, generated with proteins found differentially up-regulated in taxol-treated fibroblasts, strikingly correlates with recurrence, metastasis and poor patient survival in several solid malignancies. We therefore suggest the inhibition of the catabolic state in healthy cells as a novel approach to improve current chemotherapy efficacies and possibly avoid future carcinogenic processes.
Asunto(s)
Antineoplásicos/farmacología , Azatioprina/farmacología , Biomarcadores de Tumor/metabolismo , Fibroblastos/metabolismo , Paclitaxel/farmacología , Antioxidantes/metabolismo , Autofagia , Diferenciación Celular , Células Cultivadas , Senescencia Celular , Supervivencia sin Enfermedad , Fibroblastos/efectos de los fármacos , Humanos , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/metabolismo , Proteómica , Estrés Fisiológico , Microambiente TumoralRESUMEN
Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres (i.e., stem cell activity) and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(-) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2 (cytochrome c oxidase subunit 2; Complex IV) expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells in vivo. Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Telomerasa/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Mitocondrias/genética , Proteómica/métodos , Telomerasa/genética , Regulación hacia ArribaRESUMEN
We have used an unbiased proteomic profiling strategy to identify new potential therapeutic targets in tumor-initiating cells (TICs), a.k.a., cancer stem cells (CSCs). Towards this end, the proteomes of mammospheres from two breast cancer cell lines were directly compared to attached monolayer cells. This allowed us to identify proteins that were highly over-expressed in CSCs and/or progenitor cells. We focused on ribosomal proteins and protein folding chaperones, since they were markedly over-expressed in mammospheres. Overall, we identified >80 molecules specifically associated with protein synthesis that were commonly upregulated in mammospheres. Most of these proteins were also transcriptionally upregulated in human breast cancer cells in vivo, providing evidence for their potential clinical relevance. As such, increased mRNA translation could provide a novel mechanism for enhancing the proliferative clonal expansion of TICs. The proteomic findings were functionally validated using known inhibitors of protein synthesis, via three independent approaches. For example, puromycin (which mimics the structure of tRNAs and competitively inhibits protein synthesis) preferentially targeted CSCs in both mammospheres and monolayer cultures, and was ~10-fold more potent for eradicating TICs, than "bulk" cancer cells. In addition, rapamycin, which inhibits mTOR and hence protein synthesis, was very effective at reducing mammosphere formation, at nanomolar concentrations. Finally, mammosphere formation was also markedly inhibited by methionine restriction, which mimics the positive effects of caloric restriction in cultured cells. Remarkably, mammosphere formation was >18-fold more sensitive to methionine restriction and replacement, as directly compared to monolayer cell proliferation. Methionine is absolutely required for protein synthesis, since every protein sequence starts with a methionine residue. Thus, the proliferation and survival of CSCs is very sensitive to the inhibition of protein synthesis, using multiple independent approaches. Our findings have important clinical implications, since they may also explain the positive therapeutic effects of PI3-kinase inhibitors and AKT inhibitors, as they ultimately converge on mTOR signaling and would block protein synthesis. We conclude that inhibition of mRNA translation by pharmacological or protein/methionine restriction may be effective strategies for eliminating TICs. Our data also indicate a novel mechanism by which caloric/protein restriction may reduce tumor growth, by targeting protein synthesis in anabolic tumor-initiating cancer cells.
Asunto(s)
Biomimética , Restricción Calórica , Células Madre Neoplásicas/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Inmunosupresores/farmacología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Puromicina/farmacología , Sirolimus/farmacología , Células Tumorales CultivadasRESUMEN
Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFß-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/ß-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a 'safe' mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse.
Asunto(s)
Proliferación Celular/fisiología , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Biogénesis de Organelos , Acetilcarnitina/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cromatografía Liquida , Doxiciclina/farmacología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Células MCF-7 , Mitocondrias/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Nitrilos/farmacología , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Proteómica/métodos , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Espectrometría de Masas en Tándem , Tiazoles/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved DNA-PK inhibitors have emerged, despite many years of drug discovery and lead optimization. This is largely because existing DNA-PK inhibitors suffer from poor pharmacokinetics. They are not well absorbed and/or are unstable, with a short plasma half-life. Here, we identified the first FDA-approved DNA-PK inhibitor by "chemical proteomics". In an effort to understand how doxycycline targets cancer stem-like cells (CSCs), we serendipitously discovered that doxycycline reduces DNA-PK protein expression by nearly 15-fold (> 90%). In accordance with these observations, we show that doxycycline functionally radio-sensitizes breast CSCs, by up to 4.5-fold. Moreover, we demonstrate that DNA-PK is highly over-expressed in both MCF7- and T47D-derived mammospheres. Interestingly, genetic or pharmacological inhibition of DNA-PK in MCF7 cells is sufficient to functionally block mammosphere formation. Thus, it appears that active DNA-repair is required for the clonal expansion of CSCs. Mechanistically, doxycycline treatment dramatically reduced the oxidative mitochondrial capacity and the glycolytic activity of cancer cells, consistent with previous studies linking DNA-PK expression to the proper maintenance of mitochondrial DNA integrity and copy number. Using a luciferase-based assay, we observed that doxycycline treatment quantitatively reduces the anti-oxidant response (NRF1/2) and effectively blocks signaling along multiple independent pathways normally associated with stem cells, including STAT1/3, Sonic Hedgehog (Shh), Notch, WNT and TGF-beta signaling. In conclusion, we propose that the efficacy of doxycycline as a DNA-PK inhibitor should be tested in Phase-II clinical trials, in combination with radio-therapy. Doxycycline has excellent pharmacokinetics, with nearly 100% oral absorption and a long serum half-life (18-22 hours), at a standard dose of 200-mg per day. In further support of this idea, we show that doxycycline effectively inhibits the mammosphere-forming activity of primary breast cancer samples, derived from metastatic disease sites (pleural effusions or ascites fluid). Our results also have possible implications for the radio-therapy of brain tumors and/or brain metastases, as doxycycline is known to effectively cross the blood-brain barrier. Further studies will be needed to determine if other tetracycline family members also confer radio-sensitivity.
Asunto(s)
Neoplasias de la Mama/radioterapia , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Doxiciclina/farmacología , Células Madre Neoplásicas/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Proteómica/métodosRESUMEN
Here, we developed an isogenic cell model of "stemness" to facilitate protein biomarker discovery in breast cancer. For this purpose, we used knowledge gained previously from the study of the mouse mammary tumor virus (MMTV). MMTV initiates mammary tumorigenesis in mice by promoter insertion adjacent to two main integration sites, namely Int-1 (Wnt1) and Int-2 (Fgf3), which ultimately activates Wnt/ß-catenin signaling, driving the propagation of mammary cancer stem cells (CSCs). Thus, to develop a humanized model of MMTV signaling, we over-expressed WNT1 and FGF3 in MCF7 cells, an ER(+) human breast cancer cell line. We then validated that MCF7 cells over-expressing both WNT1 and FGF3 show a 3.5-fold increase in mammosphere formation, and that conditioned media from these cells is also sufficient to promote stem cell activity in untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably reflects increased mitochondrial biogenesis. Importantly, many of the proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells in vivo, based on the bioinformatic analysis of public gene expression datasets of laser-captured patient samples. As such, this isogenic cell model should accelerate the discovery of new biomarkers to predict clinical outcome in breast cancer, facilitating the development of personalized medicine.Finally, we used mitochondrial mass as a surrogate marker for increased mitochondrial biogenesis in untransfected MCF7 cells. As predicted, metabolic fractionation of parental MCF7 cells, via MitoTracker staining, indicated that high mitochondrial mass is a new metabolic biomarker for the enrichment of anabolic CSCs, as functionally assessed by mammosphere-forming activity. This observation has broad implications for understanding the role of mitochondrial biogenesis in the propagation of stem-like cancer cells. Technically, this general metabolic approach could be applied to any cancer type, to identify and target the mitochondrial-rich CSC population.The implications of our work for understanding the role of mitochondrial metabolism in viral oncogenesis driven by random promoter insertions are also discussed, in the context of MMTV and ALV infections.
Asunto(s)
Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/patología , Factor 3 de Crecimiento de Fibroblastos/biosíntesis , Mitocondrias/fisiología , Proteína Wnt1/biosíntesis , Medios de Cultivo Condicionados/farmacología , Femenino , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Humanos , Células MCF-7 , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/patogenicidad , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Modelos Biológicos , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/patología , Esferoides Celulares/citología , Células Tumorales Cultivadas , Vía de Señalización Wnt/fisiología , Proteína Wnt1/metabolismoRESUMEN
The abnormal haemopoietic precursor cells of chronic myeloid leukaemia (CML) carry the cytogenetic abnormality [t(9;22)(q34;q11)]--a reciprocal translocation that results in the expression of a chimaeric protein derived from the fused BCR and ABL genes. This Bcr-Abl protein tyrosine kinase mediates an array of effects on signal transduction pathways affecting cell survival, proliferation, adhesion and genetic stability. The end-result of these abnormal signalling processes is a bi- or triphasic clinical disease. Initially, CML is characterised by the presence of an excess of myeloid progenitor cells and their mature progeny. This chronic phase of CML is followed, either directly or with an intervening 'accelerated phase', by a stage where primitive blast cells predominate (acute transformation). This review discusses the role of Bcr-Abl-mediated signalling events in cellular transformation, genetic instability and disease progression in CML, and describes current developments in CML treatment using a Bcr-Abl inhibitor.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Mapeo Cromosómico , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 9 , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Genes abl/genética , Hematopoyesis , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Modelos Biológicos , Translocación GenéticaRESUMEN
Here, we used quantitative proteomics analysis to identify novel therapeutic targets in cancer stem cells and/or progenitor cells. For this purpose, mammospheres from two ER-positive breast cancer cell lines (MCF7 and T47D) were grown in suspension using low-attachment plates and directly compared to attached monolayer cells grown in parallel. This allowed us to identify a subset of proteins that were selectively over-expressed in mammospheres, relative to epithelial monolayers. We focused on mitochondrial proteins, as they appeared to be highly upregulated in both MCF7 and T47D mammospheres. Key mitochondrial-related enzymes involved in beta-oxidation and ketone metabolism were significantly upregulated in mammospheres, as well as proteins involved in mitochondrial biogenesis, and specific protein inhibitors of autophagy/mitophagy. Overall, we identified >40 "metabolic targets" that were commonly upregulated in both MCF7 and T47D mammospheres. Most of these "metabolic targets" were also transcriptionally upregulated in human breast cancer cells in vivo, validating their clinical relevance. Based on this analysis, we propose that increased mitochondrial biogenesis and decreased mitochondrial degradation could provide a novel mechanism for the accumulation of mitochondrial mass in cancer stem cells. To functionally validate our observations, we utilized a specific MCT1/2 inhibitor (AR-C155858), which blocks the cellular uptake of two types of mitochondrial fuels, namely ketone bodies and L-lactate. Our results indicate that inhibition of MCT1/2 function effectively reduces mammosphere formation, with an IC-50 of ~1 µM, in both ER-positive and ER-negative breast cancer cell lines. Very similar results were obtained with oligomycin A, an inhibitor of the mitochondrial ATP synthase. Thus, the proliferative clonal expansion of cancer stem cells appears to require oxidative mitochondrial metabolism, related to the re-use of monocarboxylic acids, such as ketones or L-lactate. Our findings have important clinical implications for exploiting mitochondrial metabolism to eradicate cancer stem cells and to prevent recurrence, metastasis and drug resistance in cancer patients. Importantly, a related MCT1/2 inhibitor (AZD3965) is currently in phase I clinical trials in patients with advanced cancers: http://clinicaltrials.gov/show/NCT01791595.