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Clinical cytogenomic studies of solid tumor samples are critical to the diagnosis, prognostication, and treatment selection for cancer patients. An overview of current cytogenomic techniques for solid tumor analysis is provided, including standards for sample preparation, clinical and technical considerations, and documentation of results. With the evolving technologies and their application in solid tumor analysis, these standards now include sequencing technology and optical genome mapping, in addition to the conventional cytogenomic methods, such as G-banded chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray analysis. This updated Section E6.7-6.12 supersedes the previous Section E6.5-6.8 in Section E: Clinical Cytogenetics of the American College of Medical Genetics and Genomics Standards for Clinical Genetics Laboratories.
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Genética Médica , Neoplasias , Humanos , Estados Unidos , Laboratórios , Hibridização in Situ Fluorescente/métodos , Aberrações Cromossômicas , Neoplasias/diagnóstico , Neoplasias/genética , Cromossomos , GenômicaRESUMO
Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for â¼30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, ≥99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 single-nucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing-based panel testing should be a part of routine diagnostics in patients with suspected IBMFS.
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Anemia Aplástica , Doenças da Medula Óssea , Hemoglobinúria Paroxística , Humanos , Criança , Anemia Aplástica/diagnóstico , Anemia Aplástica/genética , Doenças da Medula Óssea/diagnóstico , Doenças da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea , Variações do Número de Cópias de DNA/genética , Reprodutibilidade dos Testes , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , NucleotídeosRESUMO
Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the development of tumors of the nervous system and is associated with NF2 gene alterations. Atypical teratoid rhabdoid tumor (ATRT) is a malignant central nervous system tumor that occurs primarily in children less than 3 years of age. The majority of cases of ATRT demonstrate genomic alterations of SMARCB1, a core member of the SWI/SNF chromatin-remodeling complex and tumor suppressor gene. SMARCB1 inactivation in ATRT is occasionally associated with somatic NF2 deletion; however, concurrent germline NF2 mutations have not been reported. Herein, we describe the case of a 3-year-old patient who presented with an intracranial mass. Next generation sequencing analysis of tumor identified homozygous deletions of the entire SMARCB1 gene and exon 7 to exon 14 of NF2 gene with whole chromosome 22 loss of heterozygosity (LOH). Multiplex Ligation-dependent Probe Amplification (MLPA) assay performed on blood identified a germline heterozygous intragenic deletion of NF2 exon 7 to exon 14; a somatic chromosome 22 LOH led to the homozygous deletion. SMARCB1 MLPA assay of blood showed no deletion. This cascade represents a novel, "four-hit" mechanism of SMARCB1 inactivation resulting in ATRT and the first known dual diagnosis of NF2 and ATRT.
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Neoplasias do Sistema Nervoso Central , Neoplasias Neuroepiteliomatosas , Neurofibromatose 2 , Tumor Rabdoide , Teratoma , Neoplasias do Sistema Nervoso Central/genética , Pré-Escolar , Homozigoto , Humanos , Neoplasias Neuroepiteliomatosas/genética , Neurofibromatose 2/genética , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Deleção de Sequência , Teratoma/genética , Teratoma/patologiaRESUMO
The NTRK gene family is composed of NTRK1, NTRK2, and NTRK3, which encode three tropomyosin-receptor kinases, belonging to a class of tyrosine kinase receptors. These proteins are known to play roles in cell proliferation, differentiation, apoptosis, and survival. Fusions involving the NTRK genes are long known as drivers in many tumors. Although they occur in less than 5% of all malignancies, their occurrence in a great diversity of tumors has been documented. Several rare tumors including infantile fibrosarcoma, secretory breast carcinoma, and mammary analogue secretory carcinoma are accompanied by NTRK fusions in more than 90% of cases, demonstrating a diagnostic value for the NTRK fusion testing in these tumors. More recently, the development of effective targeted therapies has created a demand for their detection in all malignancies. A variety of approaches are available for testing including immunohistochemistry, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and DNA- and RNA-based next-generation sequencing (NGS). This article reviews the molecular biology and tumorigenesis of NTRK fusions, their prevalence and clinical significance with a focus on available methods for fusion detection. The advantages and limitations of different technologies, the best practice algorithms for NTRK fusion detection, and the future direction of NTRK testing are also discussed.
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Biomarcadores Tumorais/genética , Neoplasias/diagnóstico , Proteínas de Fusão Oncogênica/genética , Receptor trkA/genética , Receptor trkB/genética , Receptor trkC/genética , Animais , Humanos , Neoplasias/genética , PrognósticoRESUMO
PURPOSE: Neurotrophic tyrosine receptor kinase (NTRK) fusions have been described as oncogenic drivers in a variety of tumors. However, little is known about the overall frequency of NTRK fusion in unselected pediatric tumors. Here, we assessed the frequency, fusion partners, and clinical course in pediatric patients with NTRK fusion-positive tumors. PATIENTS AND METHODS: We studied 1,347 consecutive pediatric tumors from 1,217 patients who underwent tumor genomic profiling using custom-designed DNA and RNA next-generation sequencing panels. NTRK fusions identified were orthogonally confirmed. RESULTS AND DISCUSSION: NTRK fusions were identified in 29 tumors from 27 patients with a positive yield of 2.22% for all patients and 3.08% for solid tumors. Although NTRK2 fusions were found exclusively in CNS tumors and NTRK1 fusions were highly enriched in papillary thyroid carcinomas, NTRK3 fusions were identified in all tumor categories. The most canonical fusion was ETV6-NTRK3 observed in 10 patients with diverse types of tumors. Several novel NTRK fusions were observed in rare tumor types, including KCTD16-NTRK1 in ganglioglioma and IRF2BP2-NTRK3 in papillary thyroid carcinomas. The detection of an NTRK fusion confirmed the morphologic diagnosis including five cases where the final tumor diagnosis was largely based on the discovery of an NTRK fusion. In one patient, the diagnosis was changed because of the identification of an ETV6-NTRK3 fusion. One patient with infantile fibrosarcoma was treated with larotrectinib and achieved complete pathologic remission. CONCLUSION: NTRK fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of tumors than previously recognized. These results highlight the importance of screening for NTRK fusions as part of the tumor genomic profiling for patients with pediatric cancer.
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Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia but is approximately 500 times more likely to develop in children with Down syndrome (DS) through transformation of transient abnormal myelopoiesis (TAM). This study investigates the clinical significance of genomic heterogeneity of AMKL in children with and without DS and in children with TAM. Genomic evaluation of nine patients with DS-related TAM or AMKL, and six patients with non-DS AMKL, included conventional cytogenetics and a comprehensive next-generation sequencing panel for single-nucleotide variants/indels and copy-number variants in 118 genes and fusions involving 110 genes. Recurrent gene fusions were found in all patients with non-DS, including two individuals with complex genomes and either a NUP98-KDM5A or a KMT2A-MLLT6 fusion, and the remaining harbored a CBFA2T3-GLIS2 fusion, which arose from both typical and atypical cytogenetic mechanisms. These fusions guided treatment protocols and resulted in a change in diagnosis in two patients. The nine patients with DS had constitutional trisomy 21 and somatic GATA1 mutations, and those with DS-AMKL had two to four additional clinically significant somatic mutations. Comprehensive genomic characterization provides critical information for diagnosis, risk stratification, and treatment decisions for patients with AMKL. Continued genetic and clinical characterization of these rare cancers will aid in improving patient management.
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Genômica , Leucemia Megacarioblástica Aguda/genética , Leucemia/genética , Neoplasias/genética , Criança , Pré-Escolar , Cromossomos , Proteínas de Ligação a DNA/genética , Síndrome de Down/complicações , Síndrome de Down/genética , Feminino , Fator de Transcrição GATA1 , Predisposição Genética para Doença/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Lactente , Recém-Nascido , Cariótipo , Fatores de Transcrição Kruppel-Like/genética , Reação Leucemoide/genética , Masculino , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Proteína 2 de Ligação ao Retinoblastoma/genéticaRESUMO
ALK (Anaplastic lymphoma kinase) fusion proteins are oncogenic and have been seen in various tumors. PPP1CB-ALK fusions are rare but have been reported in a few patients with low- or high-grade gliomas. However, little is known regarding the mechanism of fusion formation and genomic break points of this fusion. We performed genomic characterization of a PPP1CB-ALK fusion with fusion gene amplification in a congenital glioblastoma. The PPP1CB-ALK consists of exons 1-5 of PPP1CB and exons 20-29 of ALK. The genomic translocation breakpoints were determined by real-time quantitative PCR (RT-qPCR) and Sanger sequencing of genomic DNA. Next generation sequencing, RT-qPCR and fluorescence in situ hybridization analyses demonstrated PPP1CB-ALK amplification. Copy number analyses of genes between PPP1CB and ALK using RT-qPCR suggest that the PPP1CB-ALK is likely the result of local chromothripsis followed by episomal amplification. Transcriptome sequencing demonstrated high-level SOX2 expression and predicted WNT/ß-catenin pathway activation, suggesting possible therapeutic approaches.
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Quinase do Linfoma Anaplásico/genética , Neoplasias Encefálicas/congênito , Amplificação de Genes , Glioblastoma/congênito , Proteína Fosfatase 1/genética , Neoplasias Encefálicas/genética , Éxons , Feminino , Glioblastoma/genética , Humanos , Recém-Nascido , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genéticaAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Histona-Lisina N-Metiltransferase/genética , Linfoma de Células B/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Adolescente , Humanos , Linfoma de Células B/genética , Masculino , PrognósticoRESUMO
Powerful, recent advances in technologies to analyze the genome have had a profound impact on the practice of medical genetics, both in the laboratory and in the clinic. Increasing utilization of genome-wide testing such as chromosomal microarray analysis and exome sequencing have lead a shift toward a "genotype-first" approach. Numerous techniques are now available to diagnose a particular syndrome or phenotype, and while traditional techniques remain efficient tools in certain situations, higher-throughput technologies have become the de facto laboratory tool for diagnosis of most conditions. However, selecting the right assay or technology is challenging, and the wrong choice may lead to prolonged time to diagnosis, or even a missed diagnosis. In this review, we will discuss current core technologies for the diagnosis of classic genetic disorders to shed light on the benefits and disadvantages of these strategies, including diagnostic efficiency, variant interpretation, and secondary findings. Finally, we review upcoming technologies posed to impart further changes in the field of genetic diagnostics as we move toward "genome-first" practice.
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PALB2 (partner and localizer of BRCA2) gene encodes a protein that colocalizes with BRCA2 in nuclear foci and likely permits the stable intranuclear localization and accumulation of BRCA2PALB2 plays a critical role in maintaining genome integrity through its role in the Fanconi anemia and homologous recombination DNA repair pathways. It has a known loss-of-function disease mechanism. Biallelic PALB2 pathogenic variants have been described in autosomal recessive Fanconi anemia. Heterozygous pathogenic variants in PALB2 are associated with increased risk for female and male breast cancer and pancreatic cancer (Science 324: 217; Cancer Res 71: 2222-2229; N Engl J Med 371: 497-506). Heterozygous germline PALB2 mutations have also been observed in patients with medulloblastoma (Lancet Oncol 19: 785-798). However, PALB2-related cancer predisposition to high-grade gliomas has not been reported. Here we report a germline PALB2 pathogenic variant (c.509_510delGA, p.Arg170Ilefs*14, NM_024675.3) found in a pediatric patient with high-grade glioma. This variant was first identified by tumor sequencing using the Children's Hospital of Philadelphia (CHOP) Comprehensive Solid Tumor Panel and then confirmed to be a germline change using the CHOP Comprehensive Hereditary Cancer Panel on DNA from a blood sample of this patient. Parental studies showed that this variant was paternally inherited. Further studies are needed to illustrate if pathogenic variants in PALB2 convey increased risk to developing brain tumor. This case also highlights the potential of identifying germline mutation through tumor sequencing.
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Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Glioma/genética , Criança , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Feminino , Predisposição Genética para Doença , Células Germinativas/metabolismo , Mutação em Linhagem Germinativa/genética , HumanosRESUMO
Gene fusions are one of the most common genomic alterations in pediatric cancer. Many fusions encode oncogenic drivers and play important roles in cancer diagnosis, risk stratification, and treatment selection. We report the development and clinical validation of a large custom-designed RNA sequencing panel, CHOP Fusion panel, using anchored multiplex PCR technology. The panel interrogates 106 cancer genes known to be involved in nearly 600 different fusions reported in hematological malignancies and solid tumors. The panel works well with different types of samples, including formalin-fixed, paraffin-embedded samples. The panel demonstrated excellent analytic accuracy, with 100% sensitivity and specificity on 60 pediatric tumor validation samples. In addition to identifying all known fusions in the validation samples, three unrecognized, yet clinically significant, fusions were also detected. A total of 276 clinical cases were analyzed after the validation, and 51 different fusions were identified in 104 cases. Of these fusions, 16 were not previously reported at the time of discovery. These fusions provided genomic information useful for clinical management. Our experience demonstrates that CHOP Fusion panel can detect the vast majority of known and certain novel clinically relevant fusion genes in pediatric cancers accurately, efficiently, and cost-effectively; and the panel provides an excellent tool for new fusion gene discovery.
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Biomarcadores Tumorais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Estudos de Casos e Controles , Criança , Genômica , Humanos , Reação em Cadeia da Polimerase MultiplexRESUMO
BACKGROUND: Somatic genetic testing is rapidly becoming the standard of care in many adult and pediatric cancers. Previously, the standard approach was single-gene or focused multigene testing, but many centers have moved towards broad-based next-generation sequencing (NGS) panels. Here, we report the laboratory validation and clinical utility of a large cohort of clinical NGS somatic sequencing results in diagnosis, prognosis, and treatment of a wide range of pediatric cancers. METHODS: Subjects were accrued retrospectively at a single pediatric quaternary-care hospital. Sequence analyses were performed on 367 pediatric cancer samples using custom-designed NGS panels over a 15-month period. Cases were profiled for mutations, copy number variations, and fusions identified through sequencing, and their clinical impact on diagnosis, prognosis, and therapy was assessed. RESULTS: NGS panel testing was incorporated meaningfully into clinical care in 88.7% of leukemia/lymphomas, 90.6% of central nervous system (CNS) tumors, and 62.6% of non-CNS solid tumors included in this cohort. A change in diagnosis as a result of testing occurred in 3.3% of cases. Additionally, 19.4% of all patients had variants requiring further evaluation for potential germline alteration. CONCLUSIONS: Use of somatic NGS panel testing resulted in a significant impact on clinical care, including diagnosis, prognosis, and treatment planning in 78.7% of pediatric patients tested in our institution. Somatic NGS tumor testing should be implemented as part of the routine diagnostic workup of newly diagnosed and relapsed pediatric cancer patients.
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DNA de Neoplasias/genética , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/diagnóstico , Análise de Sequência de DNA/métodos , Criança , DNA de Neoplasias/química , Testes Genéticos/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Neoplasias/genética , Análise de Sequência de DNA/normasAssuntos
Proteínas da Matriz do Complexo de Golgi/genética , Janus Quinase 2/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Terapia de Alvo Molecular , Nitrilas , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Pirimidinas , Indução de Remissão , Retratamento , Falha de Tratamento , Resultado do Tratamento , Proteínas de Transporte VesicularAssuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/patologia , Glioma/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Humanos , Proteínas de Filamentos Intermediários/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Binding of calcium to its intracellular receptor calmodulin (CaM) activates a family of Ca2+/CaM-dependent protein kinases. CaMKK2 (Ca2+/CaM-dependent protein kinase kinase 2) is a central member of this kinase family as it controls the actions of a CaMK cascade involving CaMKI, CaMKIV or AMPK. CaMKK2 controls insulin signaling, metabolic homeostasis, inflammation and cancer cell growth highlighting its potential as a therapeutic target for a variety of diseases. STO-609 is a selective, small molecule inhibitor of CaMKK2. Although STO-609 has been used extensively in vitro and in cells to characterize and define new mechanistic functions of CaMKK2, only a few studies have reported the in vivo use of STO-609. We synthesized functional STO-609 and assessed its pharmacological properties through in vitro (kinase assay), ex vivo (human liver microsomes) and in vivo (mouse) model systems. We describe the metabolic processing of STO-609, its toxicity, pharmacokinetics and bioavailability in a variety of mouse tissues. Utilizing these data, we show STO-609 treatment to inhibit CaMKK2 function confers protection against non-alcoholic fatty liver disease. These data provide a valuable resource by establishing criteria for use of STO-609 to inhibit the in vivo functions of CaMKK2 and demonstrate its utility for treating metabolically-related hepatic disease.
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Benzimidazóis , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Naftalimidas , Hepatopatia Gordurosa não Alcoólica , Animais , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/patologia , Naftalimidas/farmacocinética , Naftalimidas/farmacologia , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controleRESUMO
Each cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (MΦ), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as obesity and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. We developed a strategy for the simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression. To demonstrate the utility and applicability of this technique, we quantified liver cell-specific responses to high-fat diet (HFD) or diethylnitrosamine (DEN), a liver-specific carcinogen, and found that while there was only a marginal increase in hepatocyte number, MΦ and SEC populations were quantitatively increased. Global gene expression profiling of hepatocytes, MΦ and SEC identified characteristic gene signatures that define each cell type in their distinct physiological or pathological states. Integration of hepatic gene signatures with available human obesity and liver cancer microarray data provides further insight into the cell-specific responses to metabolic or oncogenic stress. Our data reveal unique gene expression patterns that serve as molecular "fingerprints" for the cell-centric responses to pathologic stimuli in the distinct microenvironment of the liver. The technical advance highlighted in this study provides an essential resource for assessing hepatic cell-specific contributions to metabolic and oncogenic stress, information that could unveil previously unappreciated molecular mechanisms for the cellular crosstalk that underlies the continuum from metabolic disruption to obesity and ultimately hepatic cancer.
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Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.
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Glucose/metabolismo , Homeostase/fisiologia , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Animais , Glucoquinase/genética , Glucoquinase/metabolismo , Camundongos , Camundongos Knockout , Transcrição GênicaRESUMO
UNLABELLED: Hepatic cancer is one of the most lethal cancers worldwide. Here, we report that the expression of Ca(2+) /calmodulin-dependent protein kinase kinase 2 (CaMKK2) is significantly up-regulated in hepatocellular carcinoma (HCC) and negatively correlated with HCC patient survival. The CaMKK2 protein is highly expressed in all eight hepatic cancer cell lines evaluated and is markedly up-regulated relative to normal primary hepatocytes. Loss of CaMKK2 function is sufficient to inhibit liver cancer cell growth, and the growth defect resulting from loss of CaMKK2 can be rescued by ectopic expression of wild-type CaMKK2 but not by kinase-inactive mutants. Cellular ablation of CaMKK2 using RNA interference yields a gene signature that correlates with improvement in HCC patient survival, and ablation or pharmacological inhibition of CaMKK2 with STO-609 impairs tumorigenicity of liver cancer cells in vivo. Moreover, CaMKK2 expression is up-regulated in a time-dependent manner in a carcinogen-induced HCC mouse model, and STO-609 treatment regresses hepatic tumor burden in this model. Mechanistically, CaMKK2 signals through Ca(2+) /calmodulin-dependent protein kinase 4 (CaMKIV) to control liver cancer cell growth. Further analysis revealed that CaMKK2 serves as a scaffold to assemble CaMKIV with key components of the mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway and thereby stimulate protein synthesis through protein phosphorylation. CONCLUSION: The CaMKK2/CaMKIV relay is an upstream regulator of the oncogenic mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway, and the importance of this CaMKK2/CaMKIV axis in HCC growth is confirmed by the potent growth inhibitory effects of genetically or pharmacologically decreasing CaMKK2 activity; collectively, these findings suggest that CaMKK2 and CaMKIV may represent potential targets for hepatic cancer.
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Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Animais , Biópsia por Agulha , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Tomografia por Emissão de Pósitrons , Taxa de Sobrevida , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Previously, we showed that loss of CaMKK2 protected mice from high-fat diet (HFD)-induced obesity and glucose intolerance. However, although pair feeding HFD to WT mice to match food consumption of CAMKK2-null mice slowed weight gain, it failed to protect from glucose intolerance. Here we show that relative to WT mice, HFD-fed CaMKK2-null mice are protected from inflammation in adipose and remain glucose-tolerant. Moreover, loss of CaMKK2 also protected mice from endotoxin shock and fulminant hepatitis. We explored the expression of CaMKK2 in immune cells and found it to be restricted to those of the monocyte/macrophage lineage. CaMKK2-null macrophages exhibited a remarkable deficiency to spread, phagocytose bacteria, and synthesize cytokines in response to the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncoupled the TLR4 cascade from activation of protein tyrosine kinase 2 (PYK2; also known as PTK2B). Our findings uncover an important function for CaMKK2 in mediating mechanisms that control the amplitude of macrophage inflammatory responses to excess nutrients or pathogen derivatives.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Macrófagos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/deficiência , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Adesão Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Dieta Hiperlipídica/efeitos adversos , Ativação Enzimática/efeitos dos fármacos , Quinase 2 de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Intolerância à Glucose/etiologia , Intolerância à Glucose/prevenção & controle , Hepatite/etiologia , Hepatite/prevenção & controle , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Choque Séptico/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca(2+)/CaM-dependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system -signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism. CaMKK2 is shown to be expressed in liver and acute, specific reduction of the kinase in the liver of high-fat diet-fed CaMKK2(floxed) mice results in lowered blood glucose and improved glucose tolerance. Primary hepatocytes isolated from CaMKK2 KO mice produce less glucose and have decreased mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α and the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and these mRNA fail to respond specifically to the stimulatory effect of catecholamine in a cell-autonomous manner. The mechanism responsible for suppressed gene induction in CaMKK2 KO hepatocytes may involve diminished phosphorylation of histone deacetylase 5, an event necessary in some contexts for derepression of the peroxisome proliferator-activated receptor γ coactivator 1-α promoter. Hepatocytes from CaMKK2 KO mice also show increased rates of de novo lipogenesis and fat oxidation. The changes in fat metabolism observed correlate with steatotic liver and altered acyl carnitine metabolomic profiles in CaMKK2 KO mice. Collectively, these results are consistent with suppressed catecholamine-induced induction of gluconeogenic gene expression in CaMKK2 KO mice that leads to improved whole-body glucose homeostasis despite the presence of increased hepatic fat content.