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1.
Gastroenterology ; 158(1): 238-252, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31585122

RESUMO

BACKGROUND & AIMS: We studied interactions among proteins of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which interact with microbes, and transforming growth factor beta (TGFB) signaling pathway, which is often altered in colorectal cancer cells. We investigated mechanisms by which CEACAM proteins inhibit TGFB signaling and alter the intestinal microbiome to promote colorectal carcinogenesis. METHODS: We collected data on DNA sequences, messenger RNA expression levels, and patient survival times from 456 colorectal adenocarcinoma cases, and a separate set of 594 samples of colorectal adenocarcinomas, in The Cancer Genome Atlas. We performed shotgun metagenomic sequencing analyses of feces from wild-type mice and mice with defects in TGFB signaling (Sptbn1+/- and Smad4+/-/Sptbn1+/-) to identify changes in microbiota composition before development of colon tumors. CEACAM protein and its mutants were overexpressed in SW480 and HCT116 colorectal cancer cell lines, which were analyzed by immunoblotting and proliferation and colony formation assays. RESULTS: In colorectal adenocarcinomas, high expression levels of genes encoding CEACAM proteins, especially CEACAM5, were associated with reduced survival times of patients. There was an inverse correlation between expression of CEACAM genes and expression of TGFB pathway genes (TGFBR1, TGFBR2, and SMAD3). In colorectal adenocarcinomas, we also found an inverse correlation between expression of genes in the TGFB signaling pathway and genes that regulate stem cell features of cells. We found mutations encoding L640I and A643T in the B3 domain of human CEACAM5 in colorectal adenocarcinomas; structural studies indicated that these mutations would alter the interaction between CEACAM5 and TGFBR1. Overexpression of these mutants in SW480 and HCT116 colorectal cancer cell lines increased their anchorage-independent growth and inhibited TGFB signaling to a greater extent than overexpression of wild-type CEACAM5, indicating that they are gain-of-function mutations. Compared with feces from wild-type mice, feces from mice with defects in TGFB signaling had increased abundance of bacterial species that have been associated with the development of colon tumors, including Clostridium septicum, and decreased amounts of beneficial bacteria, such as Bacteroides vulgatus and Parabacteroides distasonis. CONCLUSION: We found expression of CEACAMs and genes that regulate stem cell features of cells to be increased in colorectal adenocarcinomas and inversely correlated with expression of TGFB pathway genes. We found colorectal adenocarcinomas to express mutant forms of CEACAM5 that inhibit TGFB signaling and increase proliferation and colony formation. We propose that CEACAM proteins disrupt TGFB signaling, which alters the composition of the intestinal microbiome to promote colorectal carcinogenesis.


Assuntos
Antígeno Carcinoembrionário/genética , Carcinogênese/genética , Neoplasias Colorretais/genética , Microbioma Gastrointestinal/fisiologia , Transdução de Sinais/genética , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Antígeno Carcinoembrionário/metabolismo , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/mortalidade , Modelos Animais de Doenças , Fezes/microbiologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Mutação com Ganho de Função , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Metagenômica , Camundongos , Camundongos Transgênicos , Domínios Proteicos/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Esferoides Celulares , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismo
2.
Gastroenterology ; 157(6): 1630-1645.e6, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31560893

RESUMO

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.


Assuntos
Carcinoma Hepatocelular/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Recidiva Local de Neoplasia/genética , Adulto , Idoso , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Metilação de DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Seguimentos , Mutação com Ganho de Função , Heterogeneidade Genética , Hepatectomia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Hong Kong , Humanos , Fígado/patologia , Fígado/cirurgia , Cirrose Hepática/patologia , Cirrose Hepática/cirurgia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Masculino , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do Genoma
3.
PLoS Genet ; 15(8): e1008336, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31415562

RESUMO

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Regulon/genética , Staphylococcus aureus/metabolismo , Proteínas de Transporte de Cátions/genética , Mutação com Ganho de Função , Homeostase , Staphylococcus aureus/genética
4.
PLoS Genet ; 15(8): e1008259, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425501

RESUMO

Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections.


Assuntos
Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica/genética , Regulação Fúngica da Expressão Gênica , Antifúngicos/uso terapêutico , Candida glabrata/genética , Candidíase/microbiologia , Fluconazol/farmacologia , Fluconazol/uso terapêutico , Mutação com Ganho de Função , Histona Acetiltransferases/genética , Humanos , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Mutações Sintéticas Letais
5.
Nat Commun ; 10(1): 3810, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444327

RESUMO

Many domesticated crop plants have been bred for increased apical dominance, displaying greatly reduced axillary branching compared to their wild ancestors. In maize, this was achieved through selection for a gain-of-function allele of the TCP transcription factor teosinte branched1 (tb1). The mechanism for how a dominant Tb1 allele increased apical dominance, is unknown. Through ChIP seq, RNA seq, hormone and sugar measurements on 1 mm axillary bud tissue, we identify the genetic pathways putatively regulated by TB1. These include pathways regulating phytohormones such as gibberellins, abscisic acid and jasmonic acid, but surprisingly, not auxin. In addition, metabolites involved in sugar sensing such as trehalose 6-phosphate were increased. This suggests that TB1 induces bud suppression through the production of inhibitory phytohormones and by reducing sugar levels and energy balance. Interestingly, TB1 also putatively targets several other domestication loci, including teosinte glume architecture1, prol1.1/grassy tillers1, as well as itself. This places tb1 on top of the domestication hierarchy, demonstrating its critical importance during the domestication of maize from teosinte.


Assuntos
Domesticação , Regulação da Expressão Gênica de Plantas , Dormência de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genética , Ácido Abscísico/metabolismo , Alelos , Ciclopentanos/metabolismo , Metabolismo Energético/genética , Mutação com Ganho de Função , Genes de Plantas/genética , Loci Gênicos/genética , Oxilipinas/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Seleção Genética , Açúcares/metabolismo , Zea mays/metabolismo
6.
Mol Plant Microbe Interact ; 32(9): 1095-1109, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31365325

RESUMO

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.


Assuntos
Cruzamento , Resistência à Doença , Engenharia Genética , Fatores de Transcrição , Verticillium , Brassica rapa/microbiologia , Resistência à Doença/genética , Mutação com Ganho de Função , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Fatores de Transcrição/genética
7.
Nat Commun ; 10(1): 3533, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387997

RESUMO

People heterozygous for an activating mutation in protein kinase G1 (PRKG1, p.Arg177Gln) develop thoracic aortic aneurysms and dissections (TAAD) as young adults. Here we report that mice heterozygous for the mutation have a three-fold increase in basal protein kinase G (PKG) activity, and develop age-dependent aortic dilation. Prkg1R177Q/+ aortas show increased smooth muscle cell apoptosis, elastin fiber breaks, and oxidative stress compared to aortas from wild type littermates. Transverse aortic constriction (TAC)-to increase wall stress in the ascending aorta-induces severe aortic pathology and mortality from aortic rupture in young mutant mice. The free radical-neutralizing vitamin B12-analog cobinamide completely prevents age-related aortic wall degeneration, and the unrelated anti-oxidant N-acetylcysteine ameliorates TAC-induced pathology. Thus, increased basal PKG activity induces oxidative stress in the aorta, raising concern about the widespread clinical use of PKG-activating drugs. Cobinamide could be a treatment for aortic aneurysms where oxidative stress contributes to the disease, including Marfan syndrome.


Assuntos
Aneurisma Dissecante/prevenção & controle , Aneurisma da Aorta Torácica/prevenção & controle , Cobamidas/administração & dosagem , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Depuradores de Radicais Livres/administração & dosagem , Acetilcisteína/administração & dosagem , Aneurisma Dissecante/genética , Aneurisma Dissecante/patologia , Animais , Aorta Torácica/diagnóstico por imagem , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Feminino , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Masculino , Síndrome de Marfan/complicações , Síndrome de Marfan/genética , Camundongos , Camundongos Transgênicos , Miócitos de Músculo Liso , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Cultura Primária de Células
8.
Zhonghua Er Ke Za Zhi ; 57(8): 631-635, 2019 Aug 02.
Artigo em Chinês | MEDLINE | ID: mdl-31352750

RESUMO

Objective: To investigate the clinical and genotypic manifestations of X-linked neutropenia caused by gain-of-function mutation in WAS gene. Methods: The clinical history of two patients with X-linked neutropenia caused by gain-of-function mutation in WAS gene in Shenzhen Children's Hospital were analyzed."X-linked neutropenia" and "WAS mutation" were used as key words to search related literatures published from January 2000 to December 2018 in CNKI,Wanfang, and Pubmed databases. Results: The first case was male,1 year old, admitted for 1 year of neutropenia combined with 5 days of cough and 3 days of fever. Persistent neutropenia (0.1×10(9)-0.3×10(9)/L) was reported before admission and during hospitalization (0.4×10(9)-0.5×10(9)/L). The patient was treated with Ciprofloxacin, cefoperazone sulbactam and Vancomycin,and relieved from fever after 4 weeks of hospitalization,yet the neutropenia (0.1×10(9)-0.6×10(9)/L) continued after discharge. Variant in WAS gene (c.T869C (p.I290T) ) was identified, and the percentage of WAS protein on lymphocyte was 97.7%. The second case was male, 42 days old,admitted for fever and neutropenia (0.5×10(9)/L). Similarly,he relieved from fever after 4 weeks of treatment with amoxicillin sulbactam,vancomysin,meropenem,rifampin and isoniacid,yet was discharged with continued neutropenia. Variant in WAS gene (c.T881C (p.I294T)) was identified and the percentage of WAS protein on lymphocyte was 92%. Published literature reported four variants,including I290T, L270P, S272P and I294T, as the pathogenic mutation of X-linked neutropenia in 18 patients from five families. Neutropenia (0.1×10(9)-1.0×10(9)/L) were reported in 15 patients,while normal neutrophil number was found in the rest. Recurrent infection,mainly pneumonia and otitis media,was the most common clinical manifestation. Conclusions: Neutropenia is the prominent presentation in the patients with X-linked neutropenia caused by gain-of-function mutation in WAS gene, but it unnecessarily correlates with the clinical severity in terms of infection. Gene sequencing should be considered for the male patients with persistent neutropenia.


Assuntos
Cromossomos Humanos X , Mutação com Ganho de Função/genética , Neutropenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Febre/etiologia , Humanos , Lactente , Masculino , Mutação
9.
Oncogene ; 38(35): 6256-6269, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332290

RESUMO

p53 is known to play a role in iron homeostasis and is required for FDXR-mediated iron metabolism via iron regulatory protein 2 (IRP2). Interestingly, p53 is frequently mutated in tumors wherein iron is often accumulated, suggesting that mutant p53 may exert its gain of function by altering iron metabolism. In this study, we found that FDXR deficiency decreased mutant p53 expression along with altered iron metabolism in p53R270H/- MEFs and cancer cells carrying mutant p53. Consistently, we found that decreased expression of mutant p53 by FDXR deficiency inhibited mutant p53-R270H to induce carcinoma and high grade pleomorphic sarcoma in FDXR+/-; p53R270H/- mice as compared with p53R270H/- mice. Moreover, we found that like its effect on wild-type p53, loss of IRP2 increased mutant p53 expression. However, unlike its effect to suppress cell growth in cells carrying wild-type p53, loss of IRP2 promoted cell growth in cancer cells expressing mutant p53. Finally, we found that ectopic expression of IRP2 suppressed cell growth in a mutant p53-dependent manner. Together, our data indicate that mutant p53 gain-of-function can be suppressed by IRP2 and FDXR deficiency, both of which may be explored to target tumors carrying mutant p53.


Assuntos
Carcinogênese/genética , Proteína 2 Reguladora do Ferro/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/patologia , Células Cultivadas , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Mutação com Ganho de Função/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Células HCT116 , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
10.
Mol Immunol ; 114: 30-40, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31336247

RESUMO

Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.


Assuntos
Mutação com Ganho de Função/genética , Mutação de Sentido Incorreto/genética , Domínios Proteicos/genética , Fator de Transcrição STAT1/genética , Candidíase Mucocutânea Crônica/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Células Cultivadas , Citocinas/genética , Células HeLa , Heterozigoto , Humanos , Interferon gama/genética , Fosforilação/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Transcrição Genética/genética
11.
J Biosci Bioeng ; 128(6): 690-696, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31272833

RESUMO

Poly-γ-glutamic acid (γPGA) production by Bacillus subtilis is regulated by the quorum sensing system where DegQ transmits the cell density signal to a DNA-binding protein DegU. A mutation suppressing the γPGA-negative phenotype of degQ gene knock-out mutant (ΔdegQ) was identified through whole genome sequencing. The mutation conferred an amino acid substitution of Ser103 to phenylalanine (S103F) in yabJ that belongs to the highly conserved YjgF/YER057c/UK114 family. Genetic experiments including LacZ-fusion assay of γPGA synthetic operon confirmed that the suppressor mutation (yabJS103F) was responsible for the recovery of γPGA production. The yabJ itself was not essential for the γPGA production and the mutant allele enabled γPGA production of the ΔdegQ strain even in the presence of wild type yabJ. Thus, yabJS103F was a dominant positive allele. degU-lacZ fusion gene was hyper-expressed in cells carrying the yabJS103F, but disruption of yabJ did not affect the transcription level of the degU-lacZ. These observations suggested that YabJ acquired a function to stimulate expression of degU by the S103F mutation which is involved in the regulation of γPGA synthesis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Mutação com Ganho de Função , Ácido Poliglutâmico/análogos & derivados , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Ácido Poliglutâmico/biossíntese , Percepção de Quorum , Supressão Genética , Transativadores/metabolismo
12.
Genes (Basel) ; 10(7)2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31277422

RESUMO

While the genetic contributions to the predisposition of Bernese mountain dogs (BMDs) to histiocytic sarcoma (HS) remains unclear, some insights into key genetic drivers have been gained. Our group recently reported a mutation in the PTPN11 gene (E76K). We have now identified a second missense mutation in PTPN11 (G503V), and a mutation in KRAS (Q61H) present in HS cell lines. These mutations are associated with malignancies in humans, and known to be gain-of-function mutations that result in activation of the mitogen-activated protein kinase (MAPK) pathway. The goal of the present study was to evaluate the prevalence of these mutations in a large sample of HS cases from BMDs and golden retrievers, and in lymphoma cases, from a cohort of BMDs. Mutations in PTPN11 were present in HS in 41/96 (43%) BMDs, and in 3/13 (23%) golden retrievers. PTPN11 mutations E76K and G503V did not coexist in the same neoplasm. The KRAS mutation was much less frequent, with a prevalence of 3.1% (3/96). We did not identify either PTPN11 nor KRAS mutations in any of the lymphoma samples. These results point out the potential relevance of PTPN11 and KRAS mutations as activators of the oncogenic MAPK pathway for canine HS, particularly in BMDs.


Assuntos
Doenças do Cão/genética , Cães/genética , Sarcoma Histiocítico/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Feminino , Mutação com Ganho de Função , Sistema de Sinalização das MAP Quinases , Masculino
13.
Pediatr Rheumatol Online J ; 17(1): 38, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286971

RESUMO

BACKGROUND: Autosomal dominant gain of function mutations in caspase recruitment domain family member 14 (CARD14) is a rare condition associated with plaque-type psoriasis, generalized pustular psoriasis, palmoplantar pustular psoriasis and pityriasis rubra pilaris. Recently, a new CARD14 -associated phenotype defined as CAPE (CARD14-associated papulosquamous eruption) with clinical features of both psoriasis and pityriasis rubra pilaris was reported. We describe a family carrying a novel heterozygous mutation in CARD14 gene, with childhood-onset erythrodermic psoriasis requiring an unusual extremely high dose (up to 2 mg/kg every 8 weeks) of ustekinumab to achieve disease remission. CASE PRESENTATION: We describe a large family with three pairs of twins presenting a clinical phenotype characterized by childhood-onset erythrodermic psoriasis; in some family members is also reported psoriatic arthritis. The two probands presented poor clinical response to topic and systemic therapy with antihistamine, steroid, retinoids, cyclosporine and etanercept. After exclusion of the most common genes associated to autoinflammatory diseases (IL36RN, IL1RN, MVK, TNFRSF1A, NLRP3, NLRP12, MEFV, NOD2, PSMB8, PSTPIP1, LPIN2) we approached a new gene search by subjecting to Whole Exome Sequencing (WES) analysis five members of the family. A novel heterozygous mutation (c.446 T > G, leading to the missense amino acid substitution p.L149R) in the exon 4 of the CARD14 gene was identified in all affected members. Increasing dosages (up to 2 mg/kg every 8 weeks) of ustekinumab, a human monoclonal antibody targeting interleukin-12 (IL-12) and interleukin-23 (IL-23), allowed the complete control of the clinical manifestations, with an evident reduction of circulating Th17 and Th22 CD4+ T cell subsets. CONCLUSIONS: We describe the association of mutations of the CARD14 gene with an erythrodermic psoriasis pedigree, underlying the necessity to investigate CARD14 mutations in childhood-onset psoriasis cases and confirming the presence of CARD14 causative mutations also in erythrodermic psoriasis form, as recently reported. Also in pediatric age, ustekinumab represents a powerful therapeutic option for this rare condition, that is usually refractory to other treatments. In young children, high and frequent dosages allowed a complete control of the clinical manifestations without any severe side effects, with a long-term follow-up.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Fármacos Dermatológicos/uso terapêutico , Mutação com Ganho de Função/genética , Guanilato Ciclase/genética , Proteínas de Membrana/genética , Psoríase/tratamento farmacológico , Psoríase/genética , Ustekinumab/uso terapêutico , Criança , Dermatite Esfoliativa/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Gêmeos Dizigóticos , Sequenciamento Completo do Exoma
14.
Int J Mol Sci ; 20(11)2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185588

RESUMO

A syndrome of multiple paragangliomas/pheochromocytomas, somatostatinoma, and polycythemia due to somatic mosaic gain-of-function mutation of EPAS1, encoding HIF-2α, was previously described. HIF-2α has been implicated in endochondral and intramembranous ossification. Abnormal bone growth of the skull base may lead to Chiari malformation type I. We report two cases of EPAS1 gain-of-function mutation syndrome with Chiari malformation and developmental skull base anomalies. Patients were referred to the Section on Medical Endocrinology, Eunice Kennedy Shriver NICHD, NIH for evaluation of recurrent and metastatic paragangliomas or pheochromocytoma. The syndrome was confirmed genetically by identification of the functional EPAS1 gain-of-function mutation in the resected tumors and circulating leukocytes. Both patients were confirmed for characteristics of EPAS1 gain-of-function mutation syndrome by complete blood count (CBC), plasma biochemistry, and computed tomography (CT) of the abdomen and pelvis. Chiari malformation type I and abnormal bony development of the posterior fossa was found on MRI and CT of the head. The present study implicates EPAS1 mutations in abnormal posterior fossa development resulting in Chiari malformation type I.


Assuntos
Malformação de Arnold-Chiari/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Anormalidades Craniofaciais/genética , Paraganglioma/genética , Adulto , Malformação de Arnold-Chiari/diagnóstico por imagem , Malformação de Arnold-Chiari/patologia , Anormalidades Craniofaciais/diagnóstico por imagem , Anormalidades Craniofaciais/patologia , Feminino , Mutação com Ganho de Função , Humanos , Masculino , Pessoa de Meia-Idade , Paraganglioma/diagnóstico por imagem , Paraganglioma/patologia , Síndrome
15.
Rev Mal Respir ; 36(5): 583-590, 2019 May.
Artigo em Francês | MEDLINE | ID: mdl-31202602

RESUMO

The central nervous system (CNS), through carcinomatous meningitis or solid brain metastases, is the most common site of recurrence in non-small cell lung cancers (NSCLC) with activating mutations. Our retrospective study describes the population of patients with CNS metastases of NSCLC harboring activating mutation with targeted therapy (EGFR, ALK, BRAF, HER2) in 4 French regional reference hospitals. 60 patients were analyzed. The proposed treatments were heterogeneous and included combinations of chemotherapy, targeted therapy and radiotherapy±associated with topical treatments. Median overall survival following CNS metastasis in these patients was 15.8 months for meningitis carcinoma and 26 months for brain metastases. In patients with brain metastases, the addition of targeted therapy treatment allows a significant improvement in median progression free survival from 5.9 months to 10.6 months (HR 0.48 CI95 [0.24 to 0.97] P=0.035). These patients seem therefore benefit from systemic therapy and particularly targeted therapy with better survival than usual.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias do Sistema Nervoso Central/secundário , Neoplasias do Sistema Nervoso Central/terapia , Mutação com Ganho de Função , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias do Sistema Nervoso Central/genética , Progressão da Doença , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Receptor ErbB-2/genética , Estudos Retrospectivos
16.
Cells ; 8(7)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31248017

RESUMO

RHO GTPases are a class of small molecules involved in the regulation of several cellular processes that belong to the RAS GTPase superfamily. The RHO family of GTPases includes several members that are further divided into two different groups: typical and atypical. Both typical and atypical RHO GTPases are critical transducers of intracellular signaling and have been linked to human cancer. Significantly, both gain-of-function and loss-of-function mutations have been described in human tumors with contradicting roles depending on the cell context. The RAS family of GTPases that also belong to the RAS GTPase superfamily like the RHO GTPases, includes arguably the most frequently mutated genes in human cancers (K-RAS, N-RAS, and H-RAS) but has been extensively described elsewhere. This review focuses on the role of RHO family GTPases in human lymphoma initiation and progression.


Assuntos
Carcinogênese/genética , Linfoma/genética , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/genética , Progressão da Doença , Mutação com Ganho de Função , Humanos , Mutação com Perda de Função , Linfoma/patologia , Proteínas rho de Ligação ao GTP/metabolismo
17.
Nat Commun ; 10(1): 2701, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221965

RESUMO

One of the biggest hurdles for the development of metabolism-targeted therapies is to identify the responsive tumor subsets. However, the metabolic vulnerabilities for most human cancers remain unclear. Establishing the link between metabolic signatures and the oncogenic alterations of receptor tyrosine kinases (RTK), the most well-defined cancer genotypes, may precisely direct metabolic intervention to a broad patient population. By integrating metabolomics and transcriptomics, we herein show that oncogenic RTK activation causes distinct metabolic preference. Specifically, EGFR activation branches glycolysis to the serine synthesis for nucleotide biosynthesis and redox homeostasis, whereas FGFR activation recycles lactate to fuel oxidative phosphorylation for energy generation. Genetic alterations of EGFR and FGFR stratify the responsive tumors to pharmacological inhibitors that target serine synthesis and lactate fluxes, respectively. Together, this study provides the molecular link between cancer genotypes and metabolic dependency, providing basis for patient stratification in metabolism-targeted therapies.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Mutação com Ganho de Função , Perfilação da Expressão Gênica/métodos , Glicólise/efeitos dos fármacos , Glicólise/genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Seleção de Pacientes , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Serina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Int J Med Sci ; 16(5): 757-765, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217744

RESUMO

Background: The D816V mutation of c-KIT can constitutively activate tyrosine kinase, thereby promote core binding factor acute myeloid leukemia (CBF-AML) cell proliferation and inhibit apoptosis. Previous studies have indicated similar proliferation and apoptosis between N822K and D816V mutations.The current study aims to determine the occurrence and potential functions of N822K mutation-induced c-KIT activation in AML cells, and explore possible mechanisms of poor prognosis of CBF-AML. Methods: c-KIT N822K mutation status in AML cells was determined by exon 17 sequencing. The level of c-KIT expression was detected by flow cytometry (FCM) and colony formation was assessed after hu-SCF stimulation. After exposure to sunitinib (a kind of tyrosine kinase inhibitor, TKI), cell proliferation inhibition was tested by MTT, cell cycle and apoptosis were measured by FCM, autophagy was assessed by fluorescence microscopy and immunoblotting. Results: Kasumi-1 cell line was detected to bear c-KIT N822K (T>A) mutation. After hu-SCF stimulation, CD117 expression was decreased and the colony formation efficiency was not altered in Kasumi-1 cells. After sunitinib inhibited the c-KIT activity, the colony formation efficiency was reduced, and the half-maximal inhibitory concentration (IC50) of sunitinib was low (0.44±0.17µM) at 48 hours. Moreover, cells were arrested in G0/G1 phase, corresponding to an increase of apoptosis ratio. Acidic vesicular organelles (AVO) were observed along with an altered expression of autophagy-related proteins in Kasumi-1 cells. Conclusions: Our data indicated that inhibition of N822K T>A mutation-induced constitutive c-KIT activation in AML cells triggered apoptotic and autophagic pathways leading to death, and c-KIT N822K mutation may have clinical application as a CBF-AML treatment target.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Apoptose/genética , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Análise Mutacional de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Éxons/genética , Mutação com Ganho de Função , Humanos , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico
19.
Medicine (Baltimore) ; 98(18): e15329, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31045771

RESUMO

RATIONALE: Gain of function (GOF) mutations in PIK3CD gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, cytomegalovirus and/or epstein-Barr virus (EBV) viremia, and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. PATIENT CONCERNS: We report a patient who was diagnosed with systemic lupus erythematosus (SLE) at a young age and was recently found to carry heterozygous mutations in PIK3CD. The patient not only presented with recurrent sinopulmonary infections, CD4 lymphopenia, lymphadenopathy, EBV viremia, and elevated serum IgM, but also met classification criteria of SLE based on persistent proteinuria and hematuria, leukopenia and anemia, low level of serum complement, and positive autoantibody for antinuclear antibodies. DIAGNOSES: Activated PI3Kδ syndrome. INTERVENTIONS: Oral prednisolone and hydroxychloroquine combined with mycophenolate mofetil was given to the patient. He was currently receiving intravenous immunoglobulin per month in association with hydroxychloroquine, low-dose prednisolone, and mycophenolate mofetil. OUTCOMES: At present, the level of complement restored to normal, hematuria and proteinuria disappeared, and liver function returned to normal. LESSONS: SLE may be a novel phenotype of GOF mutation in PI3CKD gene (GOF PIK3CD).


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação com Ganho de Função/genética , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/genética , Lúpus Eritematoso Sistêmico/genética , Adolescente , Anticorpos Antinucleares/sangue , Grupo com Ancestrais do Continente Asiático/genética , Classe I de Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas do Sistema Complemento/análise , Proteínas do Sistema Complemento/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/imunologia , Glucocorticoides/uso terapêutico , Herpesvirus Humano 4/imunologia , Humanos , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Ácido Micofenólico/administração & dosagem , Ácido Micofenólico/uso terapêutico , Fenótipo , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico
20.
PLoS Genet ; 15(5): e1008139, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31050682

RESUMO

Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of HrasG12V, a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither HrasG12V-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espermatogônias/metabolismo , Células-Tronco Germinativas Adultas/fisiologia , Animais , Mutação com Ganho de Função/genética , Mutação em Linhagem Germinativa/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Mutação/genética , Idade Paterna , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Seleção Genética/genética , Transdução de Sinais/genética , Espermatogônias/fisiologia , Espermatozoides/metabolismo , Testículo/metabolismo
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