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1.
Cell ; 168(5): 843-855.e13, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28215706

RESUMO

The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.


Assuntos
Processamento Alternativo/efeitos da radiação , DNA Helicases/genética , RNA não Traduzido/genética , Transcrição Gênica , Raios Ultravioleta , Linhagem Celular , Éxons , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética/efeitos da radiação , Iniciação da Transcrição Genética/efeitos da radiação
2.
Cell ; 167(1): 203-218.e17, 2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27641500

RESUMO

Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7(+) γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαß(+) repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7(+) cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4(+) cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.


Assuntos
Butirofilinas/imunologia , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Butirofilinas/genética , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Timo/imunologia
3.
Immunity ; 47(6): 1083-1099.e6, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29246442

RESUMO

The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.


Assuntos
Antígeno B7-H1/imunologia , Neoplasias Colorretais/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Tristetraprolina/imunologia , Evasão Tumoral , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Tristetraprolina/genética
4.
RNA ; 29(10): 1575-1590, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37460153

RESUMO

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.


Assuntos
Fígado , Oligonucleotídeos Antissenso , Camundongos , Humanos , Animais , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Distribuição Tecidual , Fígado/diagnóstico por imagem , Fígado/metabolismo , Hibridização In Situ , Coloração e Rotulagem
5.
Anal Chem ; 95(29): 10957-10965, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37450658

RESUMO

Understanding drug fingerprints in complex biological samples is essential for the development of a drug. Hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy, a label-free nondestructive chemical imaging technique, can profile biological samples based on their endogenous vibrational contrast. Here, we propose a deep learning-assisted HS-CARS imaging approach for the investigation of drug fingerprints and their localization at single-cell resolution. To identify and localize drug fingerprints in complex biological systems, an attention-based deep neural network, hyperspectral attention net (HAN), was developed. By formulating the task to a multiple instance learning problem, HAN highlights informative regions through the attention mechanism when being trained on whole-image labels. Using the proposed technique, we investigated the drug fingerprints of a hepatitis B virus therapy in murine liver tissues. With the increase in drug dosage, higher classification accuracy was observed, with an average area under the curve (AUC) of 0.942 for the high-dose group. Besides, highly informative tissue structures predicted by HAN demonstrated a high degree of similarity with the drug localization shown by the in situ hybridization staining results. These results demonstrate the potential of the proposed deep learning-assisted optical imaging technique for the label-free profiling, identification, and localization of drug fingerprints in biological samples, which can be extended to nonperturbative investigations of complex biological systems under various biological conditions.


Assuntos
Microscopia , Análise Espectral Raman , Animais , Camundongos , Microscopia/métodos , Análise Espectral Raman/métodos , Fígado , Redes Neurais de Computação
6.
Development ; 143(10): 1674-87, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26989177

RESUMO

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Homeostase , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Pele/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dasatinibe/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Neoplasias de Células Escamosas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Cicatrização/efeitos dos fármacos , Proteínas de Sinalização YAP , Quinases da Família src/metabolismo
7.
Blood ; 129(4): 415-419, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-27903529

RESUMO

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.


Assuntos
Proteína Morfogenética Óssea 2/genética , Células Endoteliais/metabolismo , Hemocromatose/genética , Hepcidinas/genética , Homeostase/genética , Ferro/metabolismo , Fígado/metabolismo , Animais , Proteína Morfogenética Óssea 2/deficiência , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/metabolismo , Capilares/metabolismo , Capilares/patologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Hemocromatose/metabolismo , Hemocromatose/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepcidinas/metabolismo , Integrases/genética , Integrases/metabolismo , Fígado/irrigação sanguínea , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Comunicação Parácrina , Transdução de Sinais , Transcrição Gênica
8.
J Neurosci ; 37(9): 2305-2316, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28130357

RESUMO

Midbrain dopaminergic neurons are highly heterogeneous. They differ in their connectivity and firing patterns and, therefore, in their functional properties. The molecular underpinnings of this heterogeneity are largely unknown, and there is a paucity of markers that distinguish these functional subsets. In this paper, we report the identification and characterization of a novel subset of midbrain dopaminergic neurons located in the ventral tegmental area that expresses the basic helix-loop-helix transcription factor, Neurogenic Differentiation Factor-6 (NEUROD6). Retrograde fluorogold tracing experiments demonstrate that Neurod6+ midbrain dopaminergic neurons neurons project to two distinct septal regions: the dorsal and intermediate region of the lateral septum. Loss-of-function studies in mice demonstrate that Neurod6 and the closely related family member Neurod1 are both specifically required for the survival of this lateral-septum projecting neuronal subset during development. Our findings underscore the complex organization of midbrain dopaminergic neurons and provide an entry point for future studies of the functions of the Neurod6+ subset of midbrain dopaminergic neurons.SIGNIFICANCE STATEMENT Midbrain dopaminergic neurons regulate diverse brain functions, including voluntary movement and cognitive and emotive behaviors. These neurons are heterogeneous, and distinct subsets are thought to regulate different behaviors. However, we currently lack the means to identify and modify gene function in specific subsets of midbrain dopaminergic neurons. In this study, we identify the transcription factor NEUROD6 as a specific marker for a novel subset of midbrain dopaminergic neurons in the ventral midbrain that project to the lateral septum, and we reveal essential roles for Neurod1 and Neurod6 in the survival of these neurons during development. Our findings highlight the molecular and anatomical heterogeneity of midbrain dopaminergic neurons and contribute to a better understanding of this functionally complex group of neurons.


Assuntos
Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Núcleos Septais/citologia , Área Tegmentar Ventral/citologia , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biotina/análogos & derivados , Biotina/metabolismo , Calbindinas/metabolismo , Contagem de Células , Dextranos/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Vias Neurais/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Retinal Desidrogenase , Núcleos Septais/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/embriologia , Área Tegmentar Ventral/crescimento & desenvolvimento
9.
EMBO J ; 33(14): 1582-98, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-24920579

RESUMO

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endossomos/metabolismo , Modelos Biológicos , Neurônios Motores/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/genética , Imunofluorescência , Imuno-Histoquímica , Proteínas Luminescentes , Camundongos , Microscopia Eletrônica de Transmissão , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Proteína Vermelha Fluorescente
10.
Gastroenterology ; 151(3): 526-39, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27215660

RESUMO

BACKGROUND & AIMS: Pancreatitis is the most important risk factor for pancreatic ductal adenocarcinoma (PDAC). Pancreatitis predisposes to PDAC because it induces a process of acinar cell reprogramming known as acinar-to-ductal metaplasia (ADM)-a precursor of pancreatic intraepithelial neoplasia lesions that can progress to PDAC. Mutations in KRAS are found at the earliest stages of pancreatic tumorigenesis, and it appears to be a gatekeeper to cancer progression. We investigated how mutations in KRAS cooperate with pancreatitis to promote pancreatic cancer progression in mice. METHODS: We generated mice carrying conditional alleles of Yap1 and Taz and disrupted Yap1 and Taz using a Cre-lox recombination strategy in adult mouse pancreatic acinar cells (Yap1fl/fl;Tazfl/fl;Ela1-CreERT2). We crossed these mice with LSL-KrasG12D mice, which express a constitutively active form of KRAS after Cre recombination. Pancreatic tumor initiation and progression were analyzed after chemically induced pancreatitis. We analyzed pancreatic tissues from patients with pancreatitis or PDAC by immunohistochemistry. RESULTS: Oncogenic activation of KRAS in normal, untransformed acinar cells in the pancreatic tissues of mice resulted in increased levels of pancreatitis-induced ADM. Expression of the constitutive active form of KRAS in this system led to activation of the transcriptional regulators YAP1 and TAZ; their function was required for pancreatitis-induced ADM in mice. The JAK-STAT3 pathway was a downstream effector of KRAS signaling via YAP1 and TAZ. YAP1 and TAZ directly mediated transcriptional activation of several genes in the JAK-STAT3 signaling pathway; this could be a mechanism by which acinar cells that express activated KRAS become susceptible to inflammation. CONCLUSIONS: We identified a mechanism by which oncogenic KRAS facilitates ADM and thereby generates the cells that initiate neoplastic progression. This process involves activation of YAP1 and TAZ in acinar cells, which up-regulate JAK-STAT3 signaling to promote development of PDAC in mice.


Assuntos
Carcinogênese/genética , Neoplasias Pancreáticas/genética , Pancreatite/complicações , Transdução de Sinais/genética , Células Acinares/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Janus Quinases/metabolismo , Camundongos , Mutação , Pâncreas/patologia , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/patologia , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Risco , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas de Sinalização YAP
11.
J Pathol ; 237(2): 152-65, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25965974

RESUMO

Metabolic reprogramming in cancer enhances macromolecule biosynthesis and supports cell survival. Oncogenic drivers affect metabolism by altering distinct metabolic processes and render cancer cells sensitive to perturbations of the metabolic network. This study aimed to identify selective metabolic dependencies in breast cancer by investigating 17 breast cancer cells lines representative of the genetic diversity of the disease. Using a functional screen, we demonstrate here that monocarboxylate transporter 4 (MCT4) is an important regulator of breast cancer cell survival. MCT4 supports pH maintenance, lactate secretion and non-oxidative glucose metabolism in breast cancer cells. Moreover, MCT4 depletion caused an increased dependence of cancer cells on mitochondrial respiration and glutamine metabolism. MCT4 depletion reduced the ability of breast cancer cells to grow in a three-dimensional (3D) matrix or as multilayered spheroids. Moreover, MCT4 expression is regulated by the PI3K-Akt signalling pathway and highly expressed in HER2-positive breast cancers. These results suggest that MCT4 is a potential therapeutic target in defined breast cancer subtypes and reveal novel avenues for combination treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Metabolismo Energético , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , Técnicas de Cocultura , Feminino , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Células MCF-7 , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor ErbB-2/metabolismo , Transdução de Sinais , Esferoides Celulares , Fatores de Tempo , Transfecção , Carga Tumoral
12.
N Engl J Med ; 366(10): 883-892, 2012 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-22397650

RESUMO

BACKGROUND: Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS: To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS: Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS: Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).


Assuntos
Carcinoma de Células Renais/genética , Evolução Molecular , Heterogeneidade Genética , Neoplasias Renais/genética , Fenótipo , Biomarcadores Tumorais , Biópsia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/secundário , Aberrações Cromossômicas , Everolimo , Exoma , Heterogeneidade Genética/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Rim/patologia , Neoplasias Renais/patologia , Mutação , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Filogenia , Ploidias , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Sirolimo/análogos & derivados , Sirolimo/farmacologia
13.
Cancer Cell ; 11(4): 311-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17418408

RESUMO

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.


Assuntos
Carcinoma de Células Renais/etiologia , Fumarato Hidratase/genética , Inativação Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doenças Renais Císticas/etiologia , Neoplasias Renais/etiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Hipóxia Celular , Proliferação de Células , Feminino , Transportador de Glucose Tipo 1/metabolismo , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
EMBO J ; 28(13): 1843-54, 2009 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-19521338

RESUMO

Wnt signalling is a crucial signalling pathway controlling intestinal homeostasis and cancer. We show here that the JNK MAP kinase pathway and one of its most important substrates, the AP-1 transcription factor c-Jun, modulates Wnt signalling strength in the intestine. Transgenic gut-specific augmentation of JNK signalling stimulated progenitor cell proliferation and migration, resulting in increased villus length. In the crypt, c-Jun protein was highly expressed in progenitor cells and the absence of c-Jun resulted in decreased proliferation and villus length. In addition to several known c-Jun/AP-1 target genes, expression of Wnt target genes Axin2 and Lgr5 were stimulated by JNK activation, suggesting a cross talk of JNK to Wnt signalling. Expression of the Wnt pathway component TCF4 was controlled by JNK activity, and chromatin immunoprecipitation and reporter assays identified tcf4 as a direct c-Jun target gene. Consequently, increased JNK activity accelerated tumourigenesis in a model of colorectal carcinogenesis. As c-jun is a direct target of the TCF4/beta-catenin complex, the control of tcf4 expression by JNK/c-Jun leads to a positive feedback loop that connects JNK and Wnt signalling. This mechanism regulates the physiological function of progenitor cells and oncogenic transformation.


Assuntos
Carcinoma/fisiopatologia , Neoplasias do Colo/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Intestinos/citologia , Intestinos/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição TCF/genética , Fator de Transcrição 4 , Fator de Transcrição AP-1/genética , Proteínas Wnt/genética
15.
Acta Neuropathol ; 125(2): 257-72, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23179371

RESUMO

Charcot-Marie-Tooth type 2B (CMT2B) is a peripheral ulcero-mutilating neuropathy caused by four missense mutations in the rab7a gene. CMT2B is clinically characterized by prominent sensory loss, distal muscle weakness leading to muscle atrophy, high frequency of foot ulcers and infections that often results in toe amputations. RAB7A is a ubiquitous small GTPase, which controls transport to late endocytic compartments. Although the biochemical and functional properties of disease-causing RAB7A mutant proteins have been investigated, it is not yet clear how the disease originates. To understand how mutations in a ubiquitous protein specifically affect peripheral neurons, we performed a two-hybrid screen using a dorsal root ganglia cDNA library with the purpose of identifying RAB7A interactors specific for these cells. We identified peripherin, an intermediate filament protein expressed primarily in peripheral neurons, as a putative RAB7A interacting protein. The interaction was confirmed by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using recombinant proteins. Silencing or overexpression of wild type RAB7A changed the soluble/insoluble rate of peripherin indicating that RAB7A is important for peripherin organization and function. In addition, disease-causing RAB7A mutant proteins bind more strongly to peripherin and their expression causes a significant increase in the amount of soluble peripherin. Since peripherin plays a role not only in neurite outgrowth during development but also in axonal regeneration after injury, these data suggest that the altered interaction between disease-causing RAB7A mutants and peripherin could play an important role in CMT2B neuropathy.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas de Filamentos Intermediários/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Western Blotting , Células Cultivadas , Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Filamentos Intermediários/genética , Laminopatias , Glicoproteínas de Membrana/genética , Camundongos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Células PC12 , Periferinas , Plasmídeos/genética , Células do Corno Posterior/fisiologia , RNA/genética , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
16.
Stem Cells ; 30(10): 2076-87, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22851508

RESUMO

Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG-pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1-expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear ß-catenin(High) . Moreover, single NANOG1-CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem-like Wnt-associated target genes. Furthermore, we show that NANOG1-expression is positively regulated by c-JUN and ß-catenin/TCF4. Ectopic expression of c-Jun in murine Apc(Min/+) -ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c-JUN binds to the NANOG1-promoter via the octamer M1 DNA element. Collectively, our data suggest that ß-Catenin/TCF4 and c-JUN together drive a subpopulation of CRC tumor cells that adopt a stem-like phenotype via the NANOG1-promoter.


Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Sítios de Ligação , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Proteína Homeobox Nanog , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas , Ligação Proteica , Pseudogenes , Transdução de Sinais/genética , Fator de Transcrição 4 , Fatores de Transcrição/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
17.
J Pathol ; 227(2): 146-56, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22362593

RESUMO

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets. MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoural heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs. MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Glicólise/genética , Neoplasias Renais/genética , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Interferência de RNA , Apoptose , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Intervalo Livre de Doença , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Concentração de Íons de Hidrogênio , Estimativa de Kaplan-Meier , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Ácido Láctico/metabolismo , Fenótipo , Prognóstico , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção
18.
Dev Biol ; 358(1): 201-12, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21827743

RESUMO

The F-box protein Fbw7 (also known as Fbxw7, hCdc4 and Sel-10) functions as a substrate recognition component of a SCF-type E3 ubiquitin ligase. SCF(Fbw7) facilitates polyubiquitination and subsequent degradation of various proteins such as Notch, cyclin E, c-Myc and c-Jun. Fbw7 is highly expressed in the nervous system and controls neural stem cell differentiation and apoptosis via Notch and c-Jun during embryonic development (Hoeck et al., 2010). Fbw7 deletion in the neural lineage is perinatal lethal and thus prohibits studying the role of Fbw7 in the adult nervous system. fbw7 mRNA is highly expressed in the postnatal brain and to gain insights into the function of Fbw7 in postnatal neurogenesis we analysed Fbw7 function in the cerebellum. We generated conditional Fbw7-knockout mice (fbw7(∆Cb)) by inactivating Fbw7 specifically in the cerebellar anlage. This resulted in decreased cerebellar size, reduced Purkinje cell number and defects in axonal arborisation. Moreover, Fbw7-deficient cerebella showed supranumeral fissures and aberrant progenitor cell migration. Protein levels of the Fbw7 substrates Notch1 and N-terminally phosphorylated c-Jun were upregulated in fbw7(∆Cb) mice. Concomitant deletion of c-Jun, and also the junAA knock-in mutation which specifically abrogates c-Jun N-terminal phosphorylation, rescued Purkinje cell numbers and arborisation in the fbw7(∆Cb) background. Taken together these data demonstrate that Fbw7 is essential during cerebellar development, and identify N-terminally phosphorylated c-Jun as an important substrate of SCF(Fbw7) during neurogenesis.


Assuntos
Cerebelo/embriologia , Proteínas F-Box/metabolismo , Neurogênese/fisiologia , Células de Purkinje/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Southern Blotting , Cerebelo/metabolismo , Primers do DNA/genética , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-jun/metabolismo , Células de Purkinje/citologia , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética
19.
Development ; 136(23): 4043-53, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19906871

RESUMO

Sprouting angiogenesis and lymphatic-blood vessel segregation both involve the migration of endothelial cells, but the precise migratory molecules that govern the decision of blood vascular endothelial cells to segregate into lymphatic vasculature are unknown. Here, we deleted endothelial Rac1 in mice (Tie1-Cre(+);Rac1(fl/fl)) and revealed, unexpectedly, that whereas blood vessel morphology appeared normal, lymphatic-blood vessel separation was impaired, with corresponding edema, haemorrhage and embryonic lethality. Importantly, normal levels of Rac1 were essential for directed endothelial cell migratory responses to lymphatic-inductive signals. Our studies identify Rac1 as a crucial part of the migratory machinery required for endothelial cells to separate and form lymphatic vasculature.


Assuntos
Vasos Sanguíneos/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vasos Linfáticos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Separação Celular/métodos , Células Cultivadas , Embrião de Mamíferos , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Galactosídeos/metabolismo , Deleção de Genes , Imuno-Histoquímica , Indóis/metabolismo , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , RNA Interferente Pequeno/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Transfecção , beta-Galactosidase/metabolismo , Proteínas rac1 de Ligação ao GTP/análise , Proteínas rac1 de Ligação ao GTP/genética
20.
J Pathol ; 224(2): 180-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21503901

RESUMO

FBXW7 is the substrate recognition component of a SCF-type E3 ubiquitin ligase. It has multiple targets such as Notch1, c-Jun, and cyclin E that function in critical developmental and signalling pathways. Mutations in FBXW7 are often found in many types of cancer. In most cases, these mutations do not inactivate the protein, but are mono-allelic missense changes at specific arginine resides involved in substrate binding. We have hypothesized that FBXW7 mutations are selected in cancers for reasons other than haploinsufficiency or full loss-of-function. Given that the existing mutant Fbxw7 mice carry null alleles, we created a mouse model carrying one of the commonly occurring point mutations (Fbxw7(R482Q)) in the WD40 substrate recognition domain of Fbxw7. Mice heterozygous for this mutation apparently developed normally in utero, died perinatally due to a defect in lung development, and in some cases showed cleft palate and eyelid fusion defects. By comparison, Fbxw7(+/-) mice were viable and developed normally. Fbxw7(-/-) animals died of vascular abnormalities at E10.5. We screened known FBXW7 targets for changes in the lungs of the Fbxw7(R482Q/+) mice and found Tgif1 and Klf5 to be up-regulated. Fbxw7(R482Q) alleles are not functionally equivalent to heterozygous or homozygous null alleles, and we propose that they are selected in tumourigenesis because they cause a selective or partial loss of FBXW7 function.


Assuntos
Proteínas F-Box/genética , Pulmão/anormalidades , Neoplasias/genética , Mutação Puntual , Ubiquitina-Proteína Ligases/genética , Anormalidades Múltiplas/genética , Alelos , Sequência de Aminoácidos , Animais , Arginina/análise , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Proteína 7 com Repetições F-Box-WD , Humanos , Pulmão/embriologia , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Alvéolos Pulmonares/patologia , Alinhamento de Sequência , Fator de Crescimento Transformador beta/fisiologia
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