RESUMEN
Inhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)-the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM.
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Epigénesis Genética , Glioblastoma , Factores de Transcripción , Proteína p53 Supresora de Tumor , Adulto , Humanos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Histonas/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proliferación CelularRESUMEN
SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.
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Cardiolipinas , NAD , Cardiolipinas/metabolismo , Ligandos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , HumanosRESUMEN
BACKGROUND: Alzheimer's disease (AD) features progressive neurodegeneration and microglial activation that results in dementia and cognitive decline. The release of soluble amyloid (Aß) oligomers into the extracellular space is an early feature of AD pathology. This can promote excitotoxicity and microglial activation. Microglia can adopt several activation states with various functional outcomes. Protective microglial activation states have been identified in response to Aß plaque pathology in vivo. However, the role of microglia and immune mediators in neurotoxicity induced by soluble Aß oligomers is unclear. Further, there remains a need to identify druggable molecular targets that promote protective microglial states to slow or prevent the progression of AD. METHODS: Hippocampal entorhinal brain slice culture (HEBSC) was employed to study mechanisms of Aß1-42 oligomer-induced neurotoxicity as well as the role of microglia. The roles of glutamate hyperexcitation and immune signaling in Aß-induced neurotoxicity were assessed using MK801 and neutralizing antibodies to the TNF-related apoptosis-inducing ligand (TRAIL) respectively. Microglial activation state was manipulated using Gi-hM4di designer receptor exclusively activated by designer drugs (DREADDs), microglial depletion with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX3397, and microglial repopulation (PLX3397 withdrawal). Proteomic changes were assessed by LC-MS/MS in microglia isolated from control, repopulated, or Aß-treated HEBSCs. RESULTS: Neurotoxicity induced by soluble Aß1-42 oligomers involves glutamatergic hyperexcitation caused by the proinflammatory mediator and death receptor ligand TRAIL. Microglia were found to have the ability to both promote and restrain Aß-induced toxicity. Induction of microglial Gi-signaling with hM4di to prevent pro-inflammatory activation blunted Aß neurotoxicity, while microglial depletion with CSF1R antagonism worsened neurotoxicity caused by Aß as well as TRAIL. HEBSCs with repopulated microglia, however, showed a near complete resistance to Aß-induced neurotoxicity. Comparison of microglial proteomes revealed that repopulated microglia have a baseline anti-inflammatory and trophic phenotype with a predicted pathway activation that is nearly opposite that of Aß-exposed microglia. mTORC2 and IRF7 were identified as potential targets for intervention. CONCLUSION: Microglia are key mediators of both protection and neurodegeneration in response to Aß. Polarizing microglia toward a protective state could be used as a preventative strategy against Aß-induced neurotoxicity.
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Péptidos beta-Amiloides , Microglía , Fragmentos de Péptidos , Ligando Inductor de Apoptosis Relacionado con TNF , Microglía/metabolismo , Microglía/efectos de los fármacos , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/metabolismo , Animales , Fragmentos de Péptidos/toxicidad , Fragmentos de Péptidos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/toxicidad , Ratones , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Ratones Endogámicos C57BL , Corteza Entorrinal/metabolismo , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/patología , Técnicas de Cultivo de ÓrganosRESUMEN
Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success inâ vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters inâ vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46â fold) in titer and the fastest kcat (44â fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.
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Compuestos de Boro , Cloranfenicol/análogos & derivados , Escherichia coli , Pironas , Escherichia coli/genética , Catálisis , Biocatálisis , Espectrometría de MasasRESUMEN
Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.
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Factor 1 de Ribosilacion-ADP , Mapas de Interacción de Proteínas , Proteína Asociada al mTOR Insensible a la Rapamicina , Serina-Treonina Quinasas TOR , Humanos , Factor 1 de Ribosilacion-ADP/metabolismo , Insulina/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Mapeo de Interacción de Proteínas/métodosRESUMEN
Imaging defined aspects of functional tumor biology with bioluminescent reporter transgenes is a popular approach in preclinical drug development as it is sensitive, relatively high-throughput and low cost. However, the lack of internal controls subject functional bioluminescence to a number of unpredictable variables that reduce this powerful tool to semi-quantitative interpretation of large-scale effects. Here, we report the generation of sensitive and quantitative live reporters for two key measures of functional cancer biology and pharmacologic stress: the cell cycle and oxidative stress. We developed a two-colored readout, where two independent enzymes convert a common imaging substrate into spectrally distinguishable light. The signal intensity of one color is dependent upon the biological state, whereas the other color is constitutively expressed. The ratio of emitted colored light corrects the functional signal for independent procedural variables, substantially improving the robustness and interpretation of relatively low-fold changes in functional signal intensity after drug treatment. The application of these readouts in vitro is highly advantageous, as peak cell response to therapy can now be readily visualized for single or combination treatments and not simply assessed at an arbitrary and destructive timepoint. Spectral imaging in vivo can be challenging, but we also present evidence to show that the reporters can work in this context as well. Collectively, the development and validation of these internally controlled reporters allow researchers to robustly and dynamically visualize tumor cell biology in response to treatment. Given the prevalence of bioluminescence imaging, this presents significant and much needed opportunities for preclinical therapeutic development.
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Mesenchymal chondrosarcoma (MCS) is a high-grade malignancy that represents 2-9% of chondrosarcomas and mostly affects children and young adults. HEY1-NCoA2 gene fusion is considered to be a driver of tumorigenesis and it has been identified in 80% of MCS tumors. The shortage of MCS samples and biological models creates a challenge for the development of effective therapeutic strategies to improve the low survival rate of MCS patients. Previous molecular studies using immunohistochemical staining of patient samples suggest that activation of PDGFR signaling could be involved in MCS tumorigenesis. This work presents the development of two independent in vitro and in vivo models of HEY1-NCoA2-driven MCS and their application in a drug repurposing strategy. The in vitro model was characterized by RNA sequencing at the single-cell level and successfully recapitulated relevant MCS features. Imatinib, as well as specific inhibitors of ABL and PDGFR, demonstrated a highly selective cytotoxic effect targeting the HEY1-NCoA2 fusion-driven cellular model. In addition, patient-derived xenograft (PDX) models of MCS harboring the HEY1-NCoA2 fusion were developed from a primary tumor and its distant metastasis. In concordance with in vitro observations, imatinib was able to significantly reduce tumor growth in MCS-PDX models. The conclusions of this study serve as preclinical results to revisit the clinical efficacy of imatinib in the treatment of HEY1-NCoA2-driven MCS.
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Neoplasias Óseas , Condrosarcoma Mesenquimal , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carcinogénesis , Proteínas de Ciclo Celular , Reposicionamiento de Medicamentos , Xenoinjertos , Humanos , Mesilato de Imatinib , Coactivador 2 del Receptor NuclearRESUMEN
A "tug-of-war" between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases.We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the "PPPome"). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.
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Dominio Catalítico , Microcistinas/farmacología , Neoplasias/enzimología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteómica/métodos , Saccharomyces cerevisiae/enzimología , Animales , Cromatografía Liquida , Células HeLa , Humanos , Células MCF-7 , Toxinas Marinas , Ratones , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development.
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Bioensayo/métodos , Ciclo Celular , Análisis de la Célula Individual/métodos , Ubiquitinación , Antimitóticos/farmacología , Automatización , Hidrocarburos Aromáticos con Puentes/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Fluorescencia , Genes Reporteros , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Mitosis/efectos de los fármacos , Paclitaxel/farmacología , Análisis Espacio-Temporal , Taxoides/farmacología , Imagen de Lapso de Tiempo , Ubiquitinación/efectos de los fármacosRESUMEN
The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.
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Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Mapas de Interacción de Proteínas/genética , Proteína p53 Supresora de Tumor/genética , Envejecimiento/genética , Apoptosis/genética , Línea Celular , Islas de CpG/genética , Daño del ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes Supresores de Tumor , Humanos , Fenotipo , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.
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Genes Reporteros , Imagen por Resonancia Magnética/métodos , Animales , Línea Celular Tumoral , Medios de Contraste/química , Femenino , Gadolinio/química , Gadolinio DTPA/química , Células HCT116 , Células HEK293 , Humanos , Aumento de la Imagen/métodos , Iones , Células MCF-7 , Ratones , Ratones SCID , Microscopía Fluorescente/métodos , Trasplante de Neoplasias , Transportadores de Anión Orgánico/metabolismo , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin α3ß1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress α3ß1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in α3ß1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of α3ß1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Moreover, Cox-2 mRNA has reduced stability in α3ß1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies α3ß1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.
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Ciclooxigenasa 2/genética , Integrina alfa3beta1/fisiología , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama , Línea Celular Tumoral , Ciclooxigenasa 2/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Intrones , Laminina/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN Helicasas , ARN Mensajero/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , TranscriptomaRESUMEN
PURPOSE: To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), using T1- and T2-weighted imaging. METHODS: Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron-loaded or manganese-loaded ferritin, were imaged using T1- and T2-weighted MRI. Mice were also implanted subcutaneously with Timd2-expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2-weighted imaging. RESULTS: Timd2-expressing cells, but not control cells, showed a large increase in both R2 and R1 in vitro following incubation with iron-loaded and manganese-loaded ferritin, respectively. Expression of Timd2 had no effect on cell viability or proliferation; however, manganese-loaded ferritin, but not iron-loaded ferritin, was toxic to Timd2-expressing cells. Timd2-expressing xenografts in vivo showed much smaller changes in R2 following injection of iron-loaded ferritin than the same cells incubated in vitro with iron-loaded ferritin. CONCLUSION: Timd2 has demonstrated potential as an MRI reporter gene, producing large increases in R2 and R1 with ferritin and manganese-loaded ferritin respectively in vitro, although more modest changes in R2 in vivo. Manganese-loaded apoferritin was not used in vivo due to the toxicity observed in vitro. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance.
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Genes Reporteros/genética , Imagen por Resonancia Magnética/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Animales , Femenino , Ferritinas/administración & dosificación , Ferritinas/química , Ferritinas/metabolismo , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Ratones , Ratones SCIDRESUMEN
PURPOSE: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. METHODS: Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using (13) C MRS measurements of the conversion of hyperpolarized [1-(13) C] pyruvate to H(13) CO3-. RESULTS: Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two-fold increase in the H(13) CO3-/[1-(13) C] pyruvate signal ratio following intravenous injection of hyperpolarized [1-(13) C] pyruvate. CONCLUSION: We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized (13) C MRS. Magn Reson Med 76:391-401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Imagen por Resonancia Magnética/métodos , Imagen Molecular/métodos , Piruvato Descarboxilasa/metabolismo , Ácido Pirúvico/farmacocinética , Proteínas Recombinantes/metabolismo , Zymomonas/enzimología , Animales , Activación Enzimática , Femenino , Genes Reporteros/fisiología , Células HEK293 , Humanos , Ratones , Ratones SCID , Proteínas Recombinantes/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución Tisular , Zymomonas/genéticaRESUMEN
The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.
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Carcinoma/genética , Carcinoma/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/fisiología , Animales , Secuencia de Bases , Sitios de Unión/genética , Vías Biosintéticas/genética , Carcinoma/patología , Línea Celular Tumoral , Proliferación Celular , Análisis por Conglomerados , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Metabolismo/genética , Metabolismo/fisiología , Ratones , Modelos Biológicos , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Elementos de Respuesta/genética , Trasplante HeterólogoRESUMEN
Genetically engineered mouse (GEM) models of cancer currently comprise the most accurate way to experimentally recapitulate the human disease in the laboratory. Given recent advances in genomics and genetic screens, however, as well as an increasing urgency for the translation of effective preclinical treatments into the clinic, there is a pressing need to make these models easier and more efficient to work with. Accordingly, we have developed a versatile lentivirus-based approach to induce tumours from somatic cells of GEMs, add or subtract gene expression and render the tumours imageable from a simple breeding stock. The vectors deliver a tamoxifen-inducible and self-inactivating Cre recombinase, conditional bioluminescent and fluorescent proteins and an shRNA component. Following the transduction of somatic cells, tumours are initiated by Cre-mediated recombination of the inherited floxed alleles. Self-inactivation of Cre expression switches on the expression of luciferase, thereby rendering the recombined cells and resulting tumours bioluminescent. We demonstrate proof of concept of this approach by inducing bioluminescent lung tumours in conditional Kras and p53 mice. We also show that a variant vector expressing shRNA alters tumour growth dynamics and the histological grade associated with the inherited genotype. This approach comprises a versatile means to induce imageable and spontaneous tumour burden in mice. The vectors can be readily customized at the bench to modify reporter readout or tumour phenotype without additional transgenic strain development or breeding. They should also be useful for inducing imageable tumours in organs other than the lung, provided that the inherited conditional genotype is sufficiently penetrant.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Vectores Genéticos , Lentivirus/genética , Neoplasias Pulmonares/genética , Transducción Genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Integrasas/genética , Integrasas/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Clasificación del Tumor , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los Resultados , Factores de Tiempo , Carga Tumoral , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The relatively recent discovery of CRISPR/Cas has led to a revolution in our ability to efficiently manipulate the genome of eukaryotic cells. We describe here a protocol that employs CRISPR technology to precisely knock-in a PET imaging reporter transgene into a specific genetic locus of interest. Resulting transcription of the targeted reporter will more accurately mimic physiologic expression of the endogenous allele than conventional approaches, and so this method has the potential to become an efficient way to generate a new generation of "gold-standard" reporter transgenes. We break down the protocol into three experimental stages: how to identify the genomic location that the reporter transgene will be inserted, how to practically insert the reporter transgene into the genome, and how to screen resultant clones for the correct targeted event.
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Sistemas CRISPR-Cas , Genoma , Sistemas CRISPR-Cas/genética , Genes Reporteros , Transgenes , Técnicas de Sustitución del Gen , Ingeniería GenéticaRESUMEN
The broad application of noninvasive imaging has transformed preclinical cancer research, providing a powerful means to measure dynamic processes in living animals. While imaging technologies are routinely used to monitor tumor growth in model systems, their greatest potential lies in their ability to answer fundamental biological questions. Here we present the broad range of potential imaging applications according to the needs of a cancer biologist with a focus on some of the common biological processes that can be used to visualize and measure. Topics include imaging metastasis; biophysical properties such as perfusion, diffusion, oxygenation, and stiffness; imaging the immune system and tumor microenvironment; and imaging tumor metabolism. We also discuss the general ability of each approach and the level of training needed to both acquire and analyze images. The overall goal is to provide a practical guide for cancer biologists interested in answering biological questions with preclinical imaging technologies.
RESUMEN
The molecular underpinnings of H igh G rade E ndometrial C arcinoma (HGEC) metastatic growth and survival are poorly understood. Here we show that ascites-derived and primary tumor HGEC cell lines in 3D spheroid culture faithfully recapitulate key features of malignant peritoneal effusion and exhibit fundamentally distinct transcriptomic, proteomic and metabolomic landscapes when compared with conventional 2D monolayers. Using genetic screening platform we identify MAPK14 (which encodes the protein kinase p38α) as a specific requirement for HGEC in spheroid culture. MAPK14 /p38α has broad roles in programing the phosphoproteome, transcriptome and metabolome of HGEC spheroids, yet has negligible impact on monolayer cultures. MAPK14 promotes tumorigenicity in vivo and is specifically required to sustain a sub-population of spheroid cells that is enriched in cancer stemness markers. Therefore, spheroid growth of HGEC activates unique biological programs, including p38α signaling, that cannot be captured using 2D culture models and are highly relevant to malignant disease pathology.