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1.
Mol Cell ; 76(6): 872-884.e5, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31606273

RESUMO

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.


Assuntos
Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Neoplasias/enzimologia , Quinases raf/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/genética , Esferoides Celulares , Quinases raf/genética , Proteínas ras/genética
2.
Oncogene ; 38(31): 5933-5941, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285551

RESUMO

Two out of 41 non-small cell lung cancer patients enrolled in a clinical study were found with a somatic CRAF mutation in their tumor, namely CRAFP261A and CRAFP207S. To our knowledge, both mutations are novel in lung cancer and CRAFP261A has not been previously reported in cancer. Expression of CRAFP261A in HEK293T cells and BEAS-2B lung epithelial cells led to increased ERK pathway activation in a dimer-dependent manner, accompanied with loss of CRAF phosphorylation at the negative regulatory S259 residue. Moreover, stable expression of CRAFP261A in mouse embryonic fibroblasts and BEAS-2B cells led to anchorage-independent growth. Consistent with a previous report, we could not observe a gain-of-function with CRAFP207S. Type II but not type I RAF inhibitors suppressed the CRAFP261A-induced ERK pathway activity in BEAS-2B cells, and combinatorial treatment with type II RAF inhibitors and a MEK inhibitor led to a stronger ERK pathway inhibition and growth arrest. Our findings suggest that the acquisition of a CRAFP261A mutation can provide oncogenic properties to cells, and that such cells are sensitive to combined MEK and type II RAF inhibitors. CRAF mutations should be diagnostically and therapeutically explored in lung and perhaps other cancers.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores
3.
PLoS Genet ; 15(6): e1008193, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31242181

RESUMO

Mechanisms for highly efficient chromosome-associated equal segregation, and for maintenance of steady state copy number, are at the heart of the evolutionary success of the 2-micron plasmid as a stable multi-copy extra-chromosomal selfish DNA element present in the yeast nucleus. The Flp site-specific recombination system housed by the plasmid, which is central to plasmid copy number maintenance, is regulated at multiple levels. Transcription of the FLP gene is fine-tuned by the repressor function of the plasmid-coded partitioning proteins Rep1 and Rep2 and their antagonist Raf1, which is also plasmid-coded. In addition, the Flp protein is regulated by the host's post-translational modification machinery. Utilizing a Flp-SUMO fusion protein, which functionally mimics naturally sumoylated Flp, we demonstrate that the modification signals ubiquitination of Flp, followed by its proteasome-mediated degradation. Furthermore, reduced binding affinity and cooperativity of the modified Flp decrease its association with the plasmid FRT (Flp recombination target) sites, and/or increase its dissociation from them. The resulting attenuation of strand cleavage and recombination events safeguards against runaway increase in plasmid copy number, which is deleterious to the host-and indirectly-to the plasmid. These results have broader relevance to potential mechanisms by which selfish genomes minimize fitness conflicts with host genomes by holding in check the extra genetic load they pose.


Assuntos
DNA Nucleotidiltransferases/genética , Sequências Repetitivas de Ácido Nucleico/genética , Proteína SUMO-1/genética , Transcrição Genética , Segregação de Cromossomos/genética , Variações do Número de Cópias de DNA/genética , Replicação do DNA/genética , Genoma Fúngico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-raf/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação/genética , Transativadores/genética
4.
Mol Cells ; 42(6): 441-447, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31250618

RESUMO

RAS gene mutations are frequently found in one third of human cancers. Affecting approximately 1 in 1,000 newborns, germline and somatic gain-of-function mutations in the components of RAS/mitogen-activated protein kinase (RAS/MAPK) pathway has been shown to cause developmental disorders, known as RASopathies. Since RAS-MAPK pathway plays essential roles in proliferation, differentiation and migration involving developmental processes, individuals with RASopathies show abnormalities in various organ systems including central nervous system. The frequently seen neurological defects are developmental delay, macrocephaly, seizures, neurocognitive deficits, and structural malformations. Some of the defects stemmed from dysregulation of molecular and cellular processes affecting early neurodevelopmental processes. In this review, we will discuss the implications of RAS-MAPK pathway components in neurodevelopmental processes and pathogenesis of RASopathies.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Proteínas ras/genética , Síndrome de Costello/genética , Displasia Ectodérmica/genética , Facies , Insuficiência de Crescimento/genética , Cardiopatias Congênitas/genética , Humanos , Recém-Nascido , Neoplasias/metabolismo , Neurofibromatose 1/genética , Nevo Sebáceo de Jadassohn/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteína SOS1/genética , Transdução de Sinais/genética
5.
Genes (Basel) ; 10(5)2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075853

RESUMO

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.


Assuntos
Proteínas Aviárias/genética , Doenças das Aves/genética , Códon sem Sentido/genética , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Galinhas/genética , Embrião não Mamífero , Plumas/anormalidades , Deformidades Congênitas dos Membros , Mutação , Síndrome , Asas de Animais/anormalidades
6.
Cell Commun Signal ; 17(1): 31, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971268

RESUMO

BACKGROUND: p21-activated kinase 1 (PAK1) plays a fundamental role in promoting the development and progression of several cancers and is a potential therapeutic target. However, the biological function and underlying mechanism of PAK1 in esophageal squamous cell carcinoma (ESCC) remain unclear. METHODS: The expression of PAK1 was detected in both ESCC cell lines and clinical samples. Cell growth was measured by MTT, focus formation and soft agar assays. Cell migration and invasion were detected by wound healing and transwell assays. Animal models of subcutaneous tumourigenicity and tail vein metastasis were performed to determine the inhibitory effect of pharmacological inhibitor IPA-3 on tumor growth and metastasis of ESCC cells. RESULTS: We found that PAK1 was frequently overexpressed in ESCC. Ectopic expression of PAK1 promoted cellular growth, colony formation and anchorage-independent growth. Overexpressing PAK1 also enhanced migration, invasion and the expression of MMP-2 and MMP-9 in ESCC cells. In contrast, silencing PAK1 by lentiviral knockdown or a specific inhibitor IPA-3 resulted in a contrary effect. Subsequent investigations revealed that Raf1/MEK1/ERK signaling pathway was involved in PAK1-mediated effect. Enhanced expression of Raf1 attenuated the inhibitory functions of PAK1 shRNA. Whereas blocking of Raf1 by shRNA or specific inhibition of MEK1 by U0126 antagonized the oncogenetic effect of PAK1 on ESCC cells. More importantly, Pharmacological inhibition of PAK1 by IPA-3 significantly suppressed tumor growth and lung metastasis of ESCC cells in vivo. CONCLUSIONS: These data support that PAK1 is an ideal target for the development of potential therapeutic drugs for ESCC patients even with metastasis.


Assuntos
Dissulfetos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Naftóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Dissulfetos/uso terapêutico , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/secundário , Humanos , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Naftóis/uso terapêutico , Metástase Neoplásica , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Interferente Pequeno/metabolismo
7.
PLoS Genet ; 15(4): e1008108, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017896

RESUMO

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.


Assuntos
Córtex Cerebral/metabolismo , Aprendizagem , Mutação , Neuroglia/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/psicologia , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Biomarcadores , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Síndrome de Noonan/metabolismo , Oligodendroglia/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo
8.
Cancer Cell ; 35(4): 573-587.e6, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30975481

RESUMO

Five-year survival for pancreatic ductal adenocarcinoma (PDAC) patients remains below 7% due to the lack of effective treatments. Here, we report that combined ablation of EGFR and c-RAF expression results in complete regression of a significant percentage of PDAC tumors driven by Kras/Trp53 mutations in genetically engineered mice. Moreover, systemic elimination of these targets induces toxicities that are well tolerated. Response to this targeted therapy correlates with transcriptional profiles that resemble those observed in human PDACs. Finally, inhibition of EGFR and c-RAF expression effectively blocked tumor progression in nine independent patient-derived xenografts carrying KRAS and TP53 mutations. These results open the door to the development of targeted therapies for PDAC patients.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nature ; 567(7749): 521-524, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30867592

RESUMO

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600 mutations3-5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600 mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600 mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.


Assuntos
Azetidinas/uso terapêutico , Transtornos Histiocíticos Malignos/tratamento farmacológico , Transtornos Histiocíticos Malignos/enzimologia , Histiocitose/tratamento farmacológico , Histiocitose/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Piperidinas/uso terapêutico , Azetidinas/farmacologia , Transtornos Histiocíticos Malignos/genética , Transtornos Histiocíticos Malignos/patologia , Histiocitose/genética , Histiocitose/patologia , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Piperidinas/farmacologia , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética
10.
Urology ; 129: 60-67, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30904638

RESUMO

OBJECTIVE: To investigate a genetic cause of neonatal testicular torsion in 2 siblings and paternal cryptorchidism in a Caucasian family without history of consanguinity, we performed whole exome sequencing. PATIENTS AND METHODS: Targeted exon/whole-exome sequencing was performed in 2 siblings with testicular torsion. Potentially pathogenic variants passing filter criteria were validated with Sanger sequencing of parents to confirm familial segregation. Additionally, immunofluorescence staining for Raf-1, pERK (downstream from Raf-1) and c-Kit was performed on a testicular biopsy on the preserved testicle from the proband brother and compared with testicular biopsies from fertile men. RESULTS: A potentially pathogenic variant was identified in the RAF1 gene (serine/threonine-protein kinase) in exon 7 of chromosome 3: 12645786 G > C; both brothers and father were heterozygous for the variant, while the mother was negative for this mutation. This mutation in exon 7 (chr3:) of RAF1 is predicted to be damaging as a highly conserved splicing site is disrupted. The mutation is not found in the single nucleotide polymorphism database, the 1000 Genomes Project, ExACT, or esp6500. Immunofluorescence of the testis biopsy from one of the brothers demonstrated markedly decreased expression of Raf-1 as well as pERK but similar expression of c-kit when compared with fertile controls. CONCLUSION: We identified a novel nonsynonymous mutation in RAF1 in n Caucasian family with testicular torsion and cryptorchidism. We present the first human evidence that the RAF/MEK/ERK pathway is associated with testicular descent.


Assuntos
Heterozigoto , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Torção do Cordão Espermático/genética , Criptorquidismo/genética , Humanos , Recém-Nascido , Masculino , Linhagem , Sequenciamento Completo do Exoma
11.
Food Chem Toxicol ; 126: 303-312, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30840849

RESUMO

With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Gö 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein ß and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein ß and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein ß and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.


Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Estaurosporina/análogos & derivados , Tretinoína/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas c-raf/genética , Estaurosporina/farmacologia
12.
Breast Cancer Res Treat ; 175(2): 317-326, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30796652

RESUMO

PURPOSE: We evaluated whether multiplex protein quantification using antibody bar-coding with photocleavable oligonucleotides (NanoString) can be applied to evaluate protein expression in breast cancer FFPE specimens. We also assessed whether diagnostic core-cuts fixed immediately at time of procedures and surgical excision sections from routinely fixed breast cancers are affected by the same fixation related differences noted using immunohistochemistry (IHC). METHODS: The expression of 26 proteins was analysed using NanoString technology in 16 pairs of FFPE breast cancer core-cuts and surgical excisions. The measurements yielded were compared with those by IHC on Ki67, PgR and HER2 biomarkers and pAKT and pERK1/2 phosphorylated proteins. RESULTS: When considered irrespective of sample type, expression measured by the two methods was strongly correlated for all markers (p < 0.001; ρ = 0.69-0.88). When core-cuts and excisions were evaluated separately, the correlations between NanoString and IHC were weaker but significant except for pAKT in excisions. Surgical excisions showed lower levels of 8/12 phosphoproteins and higher levels of 4/13 non-phosphorylated proteins in comparison to core-cuts (p < 0.01). Reduced p4EBP1, pAMPKa, pRPS6 and pRAF1 immunogenicity in excisions was correlated with tumour size and mastectomy specimens showed lower p4EBP1 and pRPS6 expression than lumpectomy (p < 0.05). CONCLUSIONS: Our study supports the validity of the new multiplex approach to protein analysis but indicates that, as with IHC, caution is necessary for the analysis in excisions particularly of phosphoproteins. The specimen type, tumour size and surgery type may lead to biases in the quantitative analysis of many proteins of biologic and clinical interest in excision specimens.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Proteínas de Neoplasias/genética , Biópsia com Agulha de Grande Calibre , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Formaldeído , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Antígeno Ki-67/genética , Mastectomia , Pessoa de Meia-Idade , Proteínas de Neoplasias/isolamento & purificação , Inclusão em Parafina , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-raf/genética , Receptor ErbB-2/genética , Receptores de Progesterona/genética
13.
Oncogene ; 38(8): 1324-1339, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30659267

RESUMO

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-RafAVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Neoplasias Mamárias Animais/genética , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Mutação , Proteínas Proto-Oncogênicas A-raf/genética , Proteínas Proto-Oncogênicas B-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/genética
14.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30610161

RESUMO

Lung cancer is the leading cause of cancer-related mortality worldwide, characterized by uncontrolled proliferation and metastasis of lung cancer cells. Tumor angiogenesis plays a key role in proliferation and metastasis in cancers, and is an essential component in microenvironment. It has been reported that long non-coding RNA FBXL19-AS1 plays an oncogenic role in colorectal cancer. However, the molecular mechanism of FBXL19-AS1 in lung cancer has not been fully elucidated. In the present study, we found that FBXL19-AS1 expression was up-regulated in lung cancer tissues and cell lines. FBXL19-AS1 knockdown inhibited cell proliferation, migration, invasion, and angiogenesis in lung cancer cells. Molecular mechanism exploration uncovered that FBXL19-AS1 acted as a molecular sponge of miR-431-5p and that RAF1 was a downstream target of miR-431-5p in lung cancer. Moreover, there was a negative association between miR-431-5p expression and FBXL19-AS1 or RAF1 expression in tumor tissues. Through rescue experiments, we discovered that overexpression of RAF1 partially rescued FBXL19-AS1 knockdown-mediated inhibition of angiogenesis and progression in lung cancer. Together, these results indicated that FBXL19-AS1 was involved in progression and angiogenesis in lung cancer by targeting miR-431-5p/RAF1 axis, which provided a new insight into the therapeutic strategies of lung cancer.


Assuntos
Neoplasias Pulmonares/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-raf/genética , RNA Longo não Codificante/genética , Células A549 , Adulto , Idoso , Carcinogênese/genética , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/patologia
15.
Hum Genet ; 138(1): 21-35, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30368668

RESUMO

RASopathies are a group of developmental disorders caused by mutations in genes that regulate the RAS/MAPK pathway and include Noonan syndrome (NS), Costello syndrome, cardiofaciocutaneous syndrome and other related disorders. Whole exome sequencing studies recently identified LZTR1, PPP1CB and MRAS as new causative genes in RASopathies. However, information on the phenotypes of LZTR1 mutation-positive patients and functional properties of the mutations are limited. To identify variants of LZTR1, PPP1CB, and MRAS, we performed a targeted next-generation sequencing and reexamined previously analyzed exome data in 166 patients with suspected RASopathies. We identified eight LZTR1 variants, including a de novo variant, in seven probands who were suspicious for NS and one known de novo PPP1CB variant in a patient with NS. One of the seven probands had two compound heterozygous LZTR1 variants, suggesting autosomal recessive inheritance. All probands with LZTR1 variants had cardiac defects, including hypertrophic cardiomyopathy and atrial septal defect. Five of the seven probands had short stature or intellectual disabilities. Immunoprecipitation of endogenous LZTR1 followed by western blotting showed that LZTR1 bound to the RAF1-PPP1CB complex. Cells transfected with a small interfering RNA against LZTR1 exhibited decreased levels of RAF1 phosphorylated at Ser259. These are the first results to demonstrate LZTR1 in association with the RAF1-PPP1CB complex as a component of the RAS/MAPK pathway.


Assuntos
Biomarcadores/análise , Mutação , Síndrome de Noonan/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Exoma , Feminino , Seguimentos , Humanos , Masculino , Síndrome de Noonan/metabolismo , Síndrome de Noonan/patologia , Fenótipo , Prognóstico , Ligação Proteica , Proteína Fosfatase 1/genética , Proteínas Proto-Oncogênicas c-raf/genética , Fatores de Transcrição/genética , Adulto Jovem
16.
J Neuropathol Exp Neurol ; 78(1): 10-14, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30517658

RESUMO

Pleomorphic xanthoastrocytoma (PXA) is a slow-growing neoplasm that predominantly affects the pediatric and young adult population. This neoplasm has a good prognosis, with a median 10-year survival rate of 70%. The majority of tumors are supratentorial and arise in the temporal lobe, while spinal tumors are extremely rare, with only 8 reported cases. Molecular perturbations involving the MAPK/ERK signaling pathway have been described in PXAs. The most common mutation is BRAF V600E in 60%-80% of cases. Other mechanisms activating this pathway in the absence of this mutation are rare and include CRAF (RAF1) fusion genes. We report a PXA case in the cervical spinal cord of a 49-year-old man with slowly progressive coordination difficulties and extremity numbness. The tumor was negative for the V600E mutation, but 2 RNA sequencing platforms detected a QKI-RAF1 fusion (t(6; 3)(q26; p25)), which has not been previously reported in PXAs. This fusion is known to activate MAPK/ERK and PI3K/mTOR signaling. Although first- and second-generation RAF inhibitors are predicted to be ineffective, this fusion may be targetable by the novel RAF inhibitor LY3009120 and to some extent by the MEK inhibitor trametinib. Genetic analysis to screen for MAPK/ERK pathway mutations is warranted on PXAs negative for the V600E mutation.


Assuntos
Astrocitoma/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas de Ligação a RNA/genética , Neoplasias da Medula Espinal/genética , Astrocitoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias da Medula Espinal/patologia
17.
Mol Carcinog ; 58(4): 554-564, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30520151

RESUMO

Emerging research has indicated that circular RNAs (circRNAs), a novel class of non-coding RNAs, play a vital role in human tumorigenesis and progression. Our previous results suggested that hsa_circ_0006528 (circ_0006528), a circRNA with an unknown function, mediates adriamycin resistance in human breast cancer cells. However, the role of circ_0006528 in breast cancer progression remains unknown. Here, we investigated the probable involvement of circ_0006528 in breast cancer. We analyzed a cohort of 97 patients and found that circ_0006528 expression was significantly upregulated in human breast cancer tissues compared with that in adjacent non-tumorous tissues and was significantly associated with advanced tumor-node-metastasis (TNM) stage and poor prognosis. In addition, we found that in breast cancer cells, circ_0006528 could promote DNA synthesis and cell proliferation, invasion, and migration. Downregulating circ_0006528 induced G2 phase arrest and cell apoptosis. Further mechanistic studies revealed that circ_0006528 could sponge endogenous miR-7-5p and inhibit its activity. We also identified Raf1, which activates the MAPK/ERK signaling pathway, as a target of miR-7-5p and determined that circ_0006528 promotes breast cancer growth, invasion, and migration by promoting the expression of Raf1 and activates the MAPK/ERK pathway. Thus, this study provides the first evidence of the circ_0006528/miR-7-5p/Raf1/MEK/ERK regulatory network in the development of breast cancer and suggests that circ_0006528 is a potential therapeutic target and prognostic predictor for breast cancer.


Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Seguimentos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Prognóstico , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais , Células Tumorais Cultivadas
18.
Eur Radiol ; 29(5): 2272-2282, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30547202

RESUMO

PURPOSE: To investigate the feasibility of prediction for targeted therapy-related gene expression in hepatocellular carcinoma (HCC) using preoperative gadoxetic acid-enhanced magnetic resonance imaging (MRI). MATERIALS AND METHODS: Ninety-one patients (81 men, mean age 53.9 ± 12 years) with solitary HCC who underwent preoperative enhanced MRI were retrospectively analyzed. Features including tumor size, signal homogeneity, tumor capsule, tumor margin, intratumoral vessels, peritumor enhancement, peritumor hypointensity, signal intensity ratio on DWI, T1 relaxation times, and the reduction rate between pre- and post-contrast enhancement images were assessed. The operation and histopathological evaluation were performed within 2 weeks after MRI examination (mean time 7 days). The expression levels of BRAF, RAF1, VEGFR2, and VEGFR3 were evaluated. The associations between these imaging features and gene expression levels were investigated. RESULTS: Tumor incomplete capsules or non-capsules (p = 0.001) and intratumoral vessels (p = 0.002) were significantly associated with BRAF expression, and tumor incomplete capsules or non-capsules (p = 0.001) and intratumoral vessels (p = 0.013) with RAF1 expression. There was no significant association between the expression of VEGFR2, VEGFR3, and all examined MRI features. Multivariate logistic regression showed that incomplete tumor capsule (p = 0.002) and non-capsule (p = 0.004) were independent risk factors of HCC with high BRAF expression; incomplete tumor capsule (p < 0.001) and non-capsule (p = 0.040) were independent risk factors of HCC with high RAF1 expression. CONCLUSION: The presence of incomplete capsule or intratumoral vessels and the absence of capsule are potential indicators of high BRAF and RAF1 expression. Gadoxetic acid-enhanced MRI may facilitate the choice of gene therapy for patients with HCC. KEY POINTS: • Incomplete tumor capsule and non-capsule were independent risk factors of HCC with high BRAF and RAF1 expression. • The presence of intratumoral vessels was a potential indicator of high BRAF and RAF1 expression. • Gadoxetic acid-enhanced MRI may be a predictor of efficacy of treatment with sorafenib.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Imagem por Ressonância Magnética , Sorafenibe/uso terapêutico , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Meios de Contraste , Estudos de Viabilidade , Feminino , Gadolínio DTPA , Humanos , Aumento da Imagem/métodos , Neoplasias Hepáticas/patologia , Imagem por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Período Pré-Operatório , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Estudos Retrospectivos , Fatores de Risco , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
19.
Fish Shellfish Immunol ; 86: 507-515, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30513386

RESUMO

RAF proto-oncogene serine/threonine-protein kinase (c-Raf) is a MAP kinase kinase kinase (MAPKKK) that participates in the Erk1/2 pathway and plays an important role in lymphocyte activation. However, the study on how c-Raf regulates adaptive immunity in non-mammal is still limited. In present study, based on analysis of sequence characteristics of c-Raf from Oreochromis niloticus (On-c-Raf), we investigated its regulation roles on teleost lymphocyte activation. The On-c-Raf was highly conserved during evolution, which was composed of a Raf-like Ras-binding domain (RBD), a protein kinase C conserved region 1 (C1) domain and a serine/threonine protein kinase catalytic (S_TKc) domain. Its mRNA showed a wide distribution in tissues of O. niloticus and with the highest expression in gill. After Aeromonas hydrophila infection, during the adaptive immune stage transcription level of On-c-Raf was significantly upregulated on day 8, but came back to original level on day 16 and 30, suggesting the potential involvement of On-c-Raf in primary response but not memory formation. Furthermore, On-c-Raf mRNA in leukocytes of Nile tilapias was obviously induced by in vitro stimulation of T cell mitogen PHA. More importantly, in vitro stimulation of lymphocytes agonist PMA augmented phosphorylation level of On-c-Raf in leukocytes detected by western-blot and immunofluorescent. Thus, c-Raf regulated lymphocyte activation of Nile tilapia on both mRNA and phosphorylation level. Together, our results revealed that the c-Raf from teleost Nile tilapia engaged in adaptive immune response by regulating lymphocytes activation. Since the regulatory mechanism of lymphocyte-mediated adaptive immunity is largely unknown in teleost, our study provided important evidences to understand teleost adaptive immunity, and also shed a novel perspective for the evolution of adaptive immune system.


Assuntos
Imunidade Adaptativa/genética , Ciclídeos/genética , Ciclídeos/imunologia , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Doenças dos Peixes/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Filogenia , Proteínas Proto-Oncogênicas c-raf/química , Alinhamento de Sequência/veterinária
20.
J Pathol ; 247(5): 539-551, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30511391

RESUMO

Melanoma is characterised by its ability to metastasise at early stages of tumour development. Current clinico-pathologic staging based on the American Joint Committee on Cancer criteria is used to guide surveillance and management in early-stage disease, but its ability to predict clinical outcome has limitations. Herein we review the genomics of melanoma subtypes including cutaneous, acral, uveal and mucosal, with a focus on the prognostic and predictive significance of key molecular aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Melanoma/genética , Mutação/genética , Neoplasias Cutâneas/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes cdc/genética , Humanos , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Neoplasias Cutâneas/patologia
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