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1.
Proc Natl Acad Sci U S A ; 117(32): 19435-19445, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719131

RESUMO

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.


Assuntos
Glutationa S-Transferase pi/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/deficiência , Glutationa S-Transferase pi/genética , Humanos , Camundongos , Camundongos Knockout , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais
2.
Ecotoxicol Environ Saf ; 196: 110476, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32278143

RESUMO

Several studies have demonstrated that PM2.5 inhalation is associated with an increased risk of cerebrovascular disease (CVD), in which inflammation plays an important role. The mechanisms of this disease are not fully understood to date. Long non-coding RNAs (lncRNAs) are involved in many pathophysiological processes, such as immune responses; however, their functions associated with inflammation are largely unexplored. High-throughput sequencing assay and obtained numerous lncRNAs that altered the expression in response to PM2.5 treatment in HUVECs. NONHSAT247851.1 was also identified, which was significantly up-regulated to control the expression of immune response genes. Mechanistically, the results indicated that NONHSAT247851.1 knockdown reduced the expression of IL1ß. In study, we investigated NONHSAT247851.1 as a promoter in regulating immune response genes via binding with raf-1 to regulate the phosphorylation level of p65 protein in HUVECs. The data collected suggests that NONHSAT247851.1 regulates inflammation via interaction with raf-1 to control the inflammatory expression in PM2.5 exposure.


Assuntos
Poluentes Ambientais/toxicidade , Inflamação/induzido quimicamente , Material Particulado/toxicidade , Proteínas Proto-Oncogênicas c-raf/genética , RNA Longo não Codificante/genética , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/genética , Interleucina-1beta/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo
3.
Ecotoxicol Environ Saf ; 194: 110412, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32155482

RESUMO

Hydrogen sulfide (H2S) is a toxic air pollutant that causes immune damage. Recent studies have found that neutrophil extracellular trap (NET) formation is one way in which neutrophils exert immune functions. In addition, the formation of NETs is also related to thrombosis and autoimmune diseases. Recent studies have shown that miRNAs are involved in the regulation of a variety of pathophysiological processes. Here, we investigated the role of H2S in regulating the formation of NETs by affecting miR-16-5p. Our study established an in vitro H2S exposure model for neutrophils using phorbol-myristate-acetate (PMA) to induce NET formation. We observed the morphological changes of cells with scanning electron microscopy and fluorescence microscopy. Then, the content of extracellular DNA and the expression of MPO and NE in each group were detected. The results showed that H2S inhibited the formation of NETs. The expression of miR-16-5p and its target genes PiK3R1 and RAF1 was then measured by qRT-PCR. H2S upregulated miR-16-5p and inhibited expression of the target genes PiK3R1 and RAF1, and it subsequently inhibited the Pi3K/AKT and ERK pathways and decreased respiratory burst levels. Furthermore, H2S attenuated inositol 1,4,5-trisphosphate receptor (IP3R)-mediated endoplasmic reticulum calcium outflow as well as autophagy caused by PMA. This study enriches H2S immunotoxicity research and provides a possible solution for the treatment of NET-related diseases.


Assuntos
Poluentes Atmosféricos/toxicidade , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Armadilhas Extracelulares/efeitos dos fármacos , Sulfeto de Hidrogênio/toxicidade , MicroRNAs/genética , Neutrófilos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Autofagia/efeitos dos fármacos , Galinhas , Armadilhas Extracelulares/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Animais , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima
4.
Proc Natl Acad Sci U S A ; 117(4): 2133-2139, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932418

RESUMO

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Consolidação da Memória , Proteínas ras/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Memória , Corpos Pedunculados/enzimologia , Neurônios/enzimologia , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/genética
5.
Future Oncol ; 16(2): 4381-4393, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31814446

RESUMO

Aim: A gene set based systematic analysis strategy is used to investigate prostate tumors and its subclusters with focuses on similarities and differences of biological functions. Results: Dysregulation of methylation status, as well as RAS/RAF/ERK and PI3K-ATK signaling pathways, were found to be the most dramatic changes during prostate cancer tumorigenesis. Besides, neural and inflammation microenvironment is also significantly divergent between tumor and adjacent tissues. Insights of subclasses within prostate tumor cohorts revealed four different clusters with distinct gene expression patterns. We found that samples are mainly clustered by immune environments and proliferation traits. Conclusion: The findings of this article may help to advance the progress of identifying better diagnosis biomarkers and therapeutic targets.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Tirosina Quinase da Agamaglobulinemia/genética , Biologia Computacional/métodos , MAP Quinases Reguladas por Sinal Extracelular/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Gradação de Tumores , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de RNA/métodos , Transdução de Sinais , Taxa de Sobrevida
6.
Int J Biochem Cell Biol ; 118: 105643, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704502

RESUMO

Colorectal cancer (CRC) is one of the most often diagnosed cancers globally. MicroRNAs are small RNA molecules that play essential roles in tumorigenesis and progression of CRC. Here we evaluated the effects of miR-141-3p on growth, cetuximab sensitivity, migration and invasion of CRC cells. We found that miR-141-3p negatively regulated the proliferation, migration and invasion in CRC cells. In addition, miR-141-3p enhanced the cetuximab sensitivity of CRC cells by EGFR suppression. Moreover, miR-141-3p improved cetuximab-induced apoptosis in CRC cells. Furthermore, miR-141-3p altered the expression of E-cadherin, N-cadherin, snail and Vimentin, indicating miR-141-3p might play a role on epithelial to mesenchymal transition (EMT). Luciferase reporter assay showed that EGFR was the direct binding site of miR-141-3p and the expression levels of p-EGFR, Raf-1, pAKT and p-ERK1/2 were regulated by miR-141-3p. After down-regulation of EGFR by siRNA in CRC cells, the effects of miR-141-3p on proliferation, migration and invasion were reversed. miR-141-3p played important roles in CRC growth and response to cetuximab treatment, and might function as a potential biomarker to predict cetuximab response.


Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , MicroRNAs/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Cetuximab/farmacologia , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-raf/genética
7.
Mol Med Rep ; 21(1): 420-428, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746389

RESUMO

Malignant melanoma has the highest malignancy rate among all skin cancer and is characterized by an insidious onset, high invasion and poor patient prognosis. Yet, the mechanisms involved remain unclear and warrant further investigation. Based on bioinformatic analysis, phospholipase C ß2 (PLCB2) has been found to be correlated with melanoma growth. The present study was the first to demonstrate that PLCB2 is a key factor affecting melanoma proliferation and apoptosis. Here, microarray datasets from the publicly available Gene Expression Omnibus (GEO) database were employed, and gene set enrichment analysis (GSEA) was introduced to identify candidate transcription factors. PLCB2 was identified as a crucial gene in the protein­protein interaction (PPI) network. The expression of PLCB2 mRNA in various cancer lines was analyzed by reverse transcription­polymerase chain reaction (RT­PCR). In addition, the proliferation ability and apoptosis rate in human melanoma cells overexpressing or not overexpressing PLCB2 were assessed using colony formation assay, flow cytometry and the Cell Counting Kit­8 (CCK­8) assay. Cell viability and apoptosis­related factors, such as p53, Bcl­2, Bax and caspase­3 were significantly regulated. Knockdown of PLCB2 suppressed the activation of the Ras/Raf/MAPK signaling pathway. In conclusion, knockdown of PLCB2 suppressed cell viability and promoted cell apoptosis by activating the Ras/Raf/MAPK pathway. Thus, PLCB2 may utilized as a potential therapeutic target in patients with melanoma.


Assuntos
Proliferação de Células/genética , Melanoma/genética , Proteínas de Neoplasias/genética , Fosfolipase C beta/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Genes ras/genética , Humanos , MAP Quinase Quinase 1/genética , Melanoma/patologia , Análise em Microsséries , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais/genética
8.
Fish Shellfish Immunol ; 96: 311-318, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31830568

RESUMO

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%-99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H2O2 treatment, in contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other hand, the total level and phosphorylation level of c-Raf significantly increased post C. irritans infection and showed an enhanced level post immunization. The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/imunologia , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Sistema Imunitário/metabolismo , Filogenia , Proteínas Proto-Oncogênicas c-raf/química , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
9.
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
10.
Oncol Rep ; 42(6): 2390-2401, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638257

RESUMO

Glioma patients receiving therapy are at a high risk of relapse and rapid progression and, thus, more effective treatments are required. The aim of the present study was to determine the suppressive role of miR­489 as an alternative therapeutic target for preventing glioma progression. The results of the present study demonstrated that patients with relatively lower levels of expression of miR­489 had more favorable clinical outcomes. Furthermore, miR­489 expression was inversely correlated with p21­activated kinase 5 (PAK5) mRNA expression levels in glioma specimens. A dual luciferase reporter assay revealed that miR­489 suppressed PAK5 expression by directly targeting the PAK5 3'­untranslated region. The effects of miR­489 on cell viability were measured using MTT and Cell Counting Kit­8 assays. The results demonstrated that ectopic expression of miR­489 mimic decreased cell viability by interfering with cyclin D1 and c­Myc signaling. Additionally, the effect of miR­489 on apoptosis was determined using Hoechst 33258 staining and flow cytometry. The results demonstrated that miR­489 decreased the activity of RAF1, reduced Bcl­2 and promoted Bax expression, resulting in increased cell apoptosis. Furthermore, the effect of miR­489 mimic on cellular motility was assessed using migration and invasion assays. miR­489 was shown to abolish the PAK5/RAF1/MMP2 pathway, resulting in decreased cell invasion ability. These results indicated that miR­489 may be involved in PAK5­mediated regulation of glioma progression, demonstrating the potential therapeutic benefits of targeting miR­489 in glioma.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Quinases Ativadas por p21/metabolismo , Biomarcadores Tumorais/genética , Proliferação de Células , Glioma/genética , Glioma/metabolismo , Humanos , Invasividade Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas c-raf/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Quinases Ativadas por p21/genética
11.
Arch Argent Pediatr ; 117(5): 330-337, 2019 10 01.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-31560489

RESUMO

INTRODUCTION: RASopathies are a set of syndromes with phenotypic overlapping features caused by gene mutations involved in the RAS/MAPK pathway. They are autosomal dominantly inherited and share common clinical characteristics, including short stature, craniofacial dysmorphisms, congenital heart disease, ectodermal manifestations, and a higher risk for cancer. A molecular diagnosis is a key factor. OBJECTIVE: To identify PTPN11, SOS1, RAF1, BRAF, and HRAS mutations and compare the main clinical characteristics of patients with molecular confirmation. Population and methods. Children with a clinical diagnosis of RASopathy assessed between August 2013 and February 2017. RESULTS: Mutations were identified in 71 % (87/122) of patients. The molecular test confirmed diagnosis in 73 % of patients with Noonan syndrome. The most prevalent mutation was c.922A>G (p.Asn308Asp) in the PTPN11 gene. A previously undescribed variant in RAF1 was detected: c.1467G>>C (p.Leu489Phe). Cardiofaciocutaneous syndrome was confirmed in 67 % of cases with BRAF mutations. Costello syndrome and Noonan syndrome with multiple lentigines were confirmed in all cases. CONCLUSION: The confirmation of clinical diagnosis allowed for a more accurate differential diagnosis. The prevalence of PTPN11 (58 %), SOS1 (10 %), and RAF1 mutations (5 %) in children with Noonan syndrome, of PTPN11 mutations (100 %) in those with Noonan syndrome with multiple lentigines, of BRAF mutations (67 %) in those with cardiofaciocutaneous syndrome, and of HRAS mutations (100 %) in those with Costello syndrome was determined.


Assuntos
Síndrome de Costello/diagnóstico , Displasia Ectodérmica/diagnóstico , Insuficiência de Crescimento/diagnóstico , Cardiopatias Congênitas/diagnóstico , Síndrome de Noonan/diagnóstico , Adolescente , Argentina , Criança , Pré-Escolar , Síndrome de Costello/genética , Diagnóstico Diferencial , Displasia Ectodérmica/genética , Facies , Insuficiência de Crescimento/genética , Feminino , Cardiopatias Congênitas/genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/genética , Adulto Jovem
12.
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
13.
Exp Dermatol ; 28(9): 1079-1082, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31338879

RESUMO

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti-cancer drugs, we generated human BRAFV600E homologous dRaf mutant (dRafA572E ) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRafA572E was expressed tissue-specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf A572E under the control of an eye-specific driver led to semi-lethality and a rough eye phenotype. The vast majority of other tissue-specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut-specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAFV600E homolog dRaf A572E exerts mostly lethal effects in Drosophila. Gut-specific dRaf A572E expression might in future be developed further for drug testing.


Assuntos
Azetidinas/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/fisiologia , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Intestinos/enzimologia , Larva , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Especificidade de Órgãos , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-raf/biossíntese , Proteínas Proto-Oncogênicas c-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/fisiologia , Vemurafenib/farmacologia
14.
Exp Mol Med ; 51(7): 70, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263178

RESUMO

Renal osteodystrophy (ROD) occurs as early as chronic kidney disease (CKD) stage 2 and seems ubiquitous in almost all pediatric patients with CKD stage 5. Fibroblast growth factor (FGF)-23, a bone-derived endocrine regulator of phosphate homeostasis, is overexpressed in CKD and disturbs osteoblast differentiation and matrix mineralization. In contrast, C-type natriuretic peptide (CNP) acts as a potent positive regulator of bone growth. In the present study, we infused CNP into uremic rats and observed whether CNP could attenuate ROD through the inhibition of FGF-23 cascades. In uremic rats, CNP administration significantly alleviated renal dysfunction, calcium phosphate metabolic disorders, hypovitaminosis D, secondary hyperparathyroidism, the decrease in bone turnover markers and retarded bone pathological progression. More importantly, within FGF-23/mitogen-activated protein kinase (MAPK) signaling, the fibroblast growth factor receptor-1, Klotho and alternative (STAT-1/phospho-STAT-1) elements were upregulated by CNP, whereas FGF-23, RAF-1/phospho-RAF-1, and downstream (ERK/phospho-ERK and P38/phospho-P38) elements were paradoxically underexpressed in bone tissue. Therefore, CNP exerts a therapeutic effect on ROD through inhibition of FGF-23/MAPK signaling at the RAF-1 level.


Assuntos
Remodelação Óssea , Distúrbio Mineral e Ósseo na Doença Renal Crônica/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Peptídeo Natriurético Tipo C/administração & dosagem , Animais , Osso e Ossos/patologia , Cálcio/sangue , Diferenciação Celular/efeitos dos fármacos , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Humanos , Rim/patologia , Masculino , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para Cima , Uremia
15.
Pediatr Transplant ; 23(6): e13535, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31259454

RESUMO

NS and related RAS/MAPK pathway (RASopathy) disorders are the leading genetic cause of HCM presenting in infancy. HCM is a major cause of morbidity and mortality in children with Noonan spectrum disorders, especially in the first year of life. Previously, there have been only isolated reports of heart transplantation as a treatment for heart failure in NS. We report on 18 patients with NS disorders who underwent heart transplantation at seven US pediatric heart transplant centers. All patients carried a NS diagnosis: 15 were diagnosed with NS and three with NSML. Sixteen of eighteen patients had comprehensive molecular genetic testing for RAS pathway mutations, with 15 having confirmed pathogenic mutations in PTPN11, RAF1, and RIT1 genes. Medical aspects of transplantation are reported as well as NS-specific medical issues. Twelve of eighteen patients described in this series were surviving at the time of data collection. Three patients died following transplantation prior to discharge from the hospital, and another three died post-discharge. Heart transplantation in NS may be a more frequent occurrence than is evident from the literature or registry data. A mortality rate of 33% is consistent with previous reports of patients with HCM transplanted in infancy and early childhood. Specific considerations may be important in evaluation of this population for heart transplant, including a potentially increased risk for malignancies as well as lymphatic, bleeding, and coagulopathy complications.


Assuntos
Insuficiência Cardíaca/cirurgia , Transplante de Coração , Síndrome de Noonan/cirurgia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/cirurgia , Pré-Escolar , Comorbidade , Feminino , Genes ras , Predisposição Genética para Doença , Insuficiência Cardíaca/genética , Humanos , Lactente , Masculino , Mutação , Síndrome de Noonan/genética , Período Pós-Operatório , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-raf/genética , Sistema de Registros , Estudos Retrospectivos , Resultado do Tratamento , Estados Unidos , Proteínas ras/genética
16.
Trends Mol Med ; 25(11): 1024-1038, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31353123

RESUMO

Inter- and intra-patient molecular heterogeneity of primary and metastatic prostate cancer (PCa) confers variable clinical outcome and poses a formidable challenge in disease management. High-throughput integrative genomics and functional approaches have untangled the complexity involved in this disease and revealed a spectrum of diverse aberrations prevalent in various molecular subtypes, including ETS fusion negative. Emerging evidence indicates that SPINK1 upregulation, mutations in epigenetic regulators or chromatin modifiers, and SPOP are associated with the ETS-fusion negative subtype. Additionally, patients with defects in a DNA-repair pathway respond to poly-(ADP-ribose)-polymerase (PARP) inhibition therapies. Furthermore, a new class of immunogenic subtype defined by CDK12 biallelic loss has also been identified in ETS-fusion-negative cases. This review focuses on the emerging molecular underpinnings driving key oncogenic aberrations and advancements in therapeutic strategies of this disease.


Assuntos
Terapia de Alvo Molecular/tendências , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias da Próstata , Proteínas Repressoras/genética , Inibidor da Tripsina Pancreática de Kazal/genética , Quinases Ciclina-Dependentes/genética , Reparo do DNA , Motivo ETS/genética , Repressão Epigenética , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Perda de Heterozigosidade , Masculino , Proteínas Nucleares/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/farmacologia , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/efeitos dos fármacos , Medicina de Precisão/tendências , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Proteômica , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Pirimidinas/uso terapêutico , Proteínas Repressoras/metabolismo , Transdução de Sinais , Inibidor da Tripsina Pancreática de Kazal/metabolismo
17.
Circulation ; 140(3): 207-224, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31163979

RESUMO

BACKGROUND: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM. METHODS: We used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control inducible pluripotent stem cell (iPSC)-derived cardiomyocytes to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease. RESULTS: We show that mutant iPSC-derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC-derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC-derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM. CONCLUSIONS: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders.


Assuntos
Cardiomiopatia Hipertrófica/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Síndrome de Noonan/genética , Proteínas Proto-Oncogênicas c-raf/genética , Adolescente , Sistemas CRISPR-Cas/fisiologia , Cardiomiopatia Hipertrófica/metabolismo , Células Cultivadas , Criança , Feminino , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Masculino , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/fisiologia , Síndrome de Noonan/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo
18.
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
19.
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
20.
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
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