RESUMEN
Hypertrophic cardiomyopathy (HCM) is the major contributor to morbidity and mortality in Noonan syndrome (NS). Gain-of-function variants in RAF1 are associated with high prevalence of HCM. Among these, NM_002880.4:c.770C > T, NP_002871.1:p.(Ser257Leu) accounts for approximately half of cases and has been reported as associated with a particularly severe outcome. Nevertheless, comprehensive studies on cases harboring this variant are missing. To precisely define the phenotype associated to the RAF1:c.770C > T, variant, an observational retrospective analysis on patients carrying the c.770C > T variant was conducted merging 17 unpublished patients and literature-derived ones. Data regarding prenatal findings, clinical features and cardiac phenotypes were collected to provide an exhaustive description of the associated phenotype. Clinical information was collected in 107 patients. Among them, 92% had HCM, mostly diagnosed within the first year of life. Thirty percent of patients were preterm and 47% of the newborns was admitted in a neonatal intensive care unit, mainly due to respiratory complications of HCM and/or pulmonary arterial hypertension. Mortality rate was 13%, mainly secondary to HCM-related complications (62%) at the average age of 7.5 months. Short stature had a prevalence of 91%, while seizures and ID of 6% and 12%, respectively. Two cases out of 75 (3%) developed neoplasms. In conclusion, patients with the RAF1:c.770C > T pathogenic variant show a particularly severe phenotype characterized by rapidly progressive neonatal HCM and high mortality rate suggesting the necessity of careful monitoring and early intervention to prevent or slow down the progression of HCM.
Asunto(s)
Cardiomiopatía Hipertrófica , Síndrome de Noonan , Fenotipo , Proteínas Proto-Oncogénicas c-raf , Humanos , Síndrome de Noonan/genética , Síndrome de Noonan/patología , Proteínas Proto-Oncogénicas c-raf/genética , Femenino , Masculino , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Lactante , Recién Nacido , Preescolar , Niño , Adolescente , Adulto , Mutación con Ganancia de FunciónRESUMEN
Nasopharyngeal carcinoma (NPC), primarily found in the southern region of China, is a malignant tumor known for its highly metastatic characteristics. The high mortality rates caused by the distant metastasis and disease recurrence remain unsolved clinical problems. In clinic, the berberine (BBR) compound has widely been in NPC therapy to decrease metastasis and disease recurrence, and BBR was documented as a main component with multiple anti-NPC effects. However, the mechanism by which BBR inhibits the growth and metastasis of nasopharyngeal carcinoma remains elusive. Herein, we show that BBR effectively inhibits the growth, metastasis, and invasion of NPC via inducing a specific super enhancer (SE). From a mechanistic perspective, the RNA sequencing (RNA-seq) results suggest that the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway, activated by the epidermal growth factor receptor (EGFR), plays a significant role in BBR-induced autophagy in NPC. Blockading of autophagy markedly attenuated the effect of BBR-mediated NPC cell growth and metastasis inhibition. Notably, BBR increased the expression of EGFR by transcription, and knockout of EGFR significantly inhibited BBR-induced microtubule associated protein 1 light chain 3 (LC3)-II increase and p62 inhibition, proposing that EGFR plays a pivotal role in BBR-induced autophagy in NPC. Chromatin immunoprecipitation sequencing (ChIP-seq) results found that a specific SE existed only in NPC cells treated with BBR. This SE knockdown markedly repressed the expression of EGFR and phosphorylated EGFR (EGFR-p) and reversed the inhibition of BBR on NPC proliferation, metastasis, and invasion. Furthermore, BBR-specific SE may trigger autophagy by enhancing EGFR gene transcription, thereby upregulating the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway. In addition, in vivo BBR effectively inhibited NPC cells growth and metastasis, following an increase LC3 and EGFR and a decrease p62. Collectively, this study identifies a novel BBR-special SE and established a new epigenetic paradigm, by which BBR regulates autophagy, inhibits proliferation, metastasis, and invasion. It provides a rationale for BBR application as the treatment regime in NPC therapy in future.
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Autofagia , Berberina , Receptores ErbB , Sistema de Señalización de MAP Quinasas , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Berberina/farmacología , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/patología , Autofagia/efectos de los fármacos , Humanos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Línea Celular Tumoral , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Animales , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proliferación Celular/efectos de los fármacos , Proteínas ras/metabolismo , Proteínas ras/genética , Ratones , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Elementos de Facilitación Genéticos/genética , Ratones DesnudosRESUMEN
The aberrant activation of RAS/RAF/MEK/ERK signaling is important for KIT mutation-mediated tumorigenesis of gastrointestinal stromal tumor (GIST). In this study, we found that inhibition of RAF1 suppresses the activation of both wild-type KIT and primary KIT mutations in GIST, with primary KIT mutations showing greater sensitivity. This suggests a positive feedback loop between KIT and RAF1, wherein RAF1 facilitates KIT signaling. We further demonstrated that RAF1 associates with KIT and the kinase activity of RAF1 is necessary for its contribution to KIT activation. Accordingly, inhibition of RAF1 suppressed cell survival, proliferation, and cell cycle progression in vitro mediated by both wild-type KIT and primary KIT mutations. Inhibition of RAF1 in vivo suppressed GIST growth in a transgenic mouse model carrying germline KIT/V558A mutation, showing a similar treatment efficiency as imatinib, the first-line targeted therapeutic drug of GIST, while the combination use of imatinib and RAF1 inhibitor further suppressed tumor growth. Acquisition of drug-resistant secondary mutation of KIT is a major cause of treatment failure of GIST following targeted therapy. Like wild-type KIT and primary KIT mutations, inhibition of RAF1 suppressed the activation of secondary KIT mutation, and the cell survival, proliferation, cell cycle progression in vitro, and tumor growth in vivo mediated by secondary KIT mutation. However, the activation of secondary KIT mutation is less dependent on RAF1 compared with that of primary KIT mutations. Taken together, our results revealed that RAF1 facilitates KIT signaling and KIT mutation-mediated tumorigenesis of GIST, providing a rationale for further investigation into the use of RAF1 inhibitors alone or in combination with KIT inhibitor in the treatment of GIST, particularly in cases resistant to KIT inhibitors.
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Tumores del Estroma Gastrointestinal , Proteínas Proto-Oncogénicas c-kit , Proteínas Proto-Oncogénicas c-raf , Transducción de Señal , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Tumores del Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Humanos , Ratones , Ratones Transgénicos , Proliferación Celular , Línea Celular Tumoral , Mutación , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Neoplasias Gastrointestinales/patología , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/metabolismoRESUMEN
Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.
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Fibrosarcoma , Nefroma Mesoblástico , Proteínas de Fusión Oncogénica , Proteínas Proto-Oncogénicas c-raf , Humanos , Fibrosarcoma/genética , Fibrosarcoma/patología , Proteínas Proto-Oncogénicas c-raf/genética , Lactante , Proteínas de Fusión Oncogénica/genética , Nefroma Mesoblástico/genética , Nefroma Mesoblástico/patología , Femenino , Masculino , Neoplasias Renales/genética , Neoplasias Renales/patología , Fusión Génica , Transducción de Señal/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proliferación Celular , Reordenamiento Génico , Proteína ETS de Variante de Translocación 6 , Receptor trkCRESUMEN
BACKGROUND: Melanoma incidence is on the rise in East Asia, yet studies of the molecular landscape are lacking in this population. We examined patients with melanoma who underwent next-generation sequencing (NGS) at a single tertiary center in South Korea, focusing on patients harboring NRAS or RAF alterations who received belvarafenib, a pan-RAF dimer inhibitor, through the Expanded Access Program (EAP). PATIENTS AND METHODS: Data were collected from 192 patients with melanoma who underwent NGS between November 2017 and May 2023. Variant call format data were obtained and annotated. Patients in the EAP received 450 mg twice daily doses of belvarafenib. RESULTS: Alterations in the RAS/RTK pathway were the most prevalent, with BRAF and NRAS alteration rates of 22.4% and 17.7%, respectively. NGS enabled additional detection of fusion mutations, including 6 BRAF and 1 RAF1 fusion. Sixteen patients with NRAS or RAF alterations received belvarafenib through the EAP, and disease control was observed in 50%, with 2 patients demonstrating remarkable responses. CONCLUSIONS: Our study highlights the value of NGS in detecting BRAF, NRAS mutations and RAF fusions, expanding possibilities for targeted therapies in malignant melanoma. Belvarafenib showed clinical benefit in patients harboring these alterations. Ongoing trials will provide further insights into the safety and efficacy of belvarafenib.
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Melanoma , Mutación , Proteínas Proto-Oncogénicas B-raf , Humanos , Melanoma/genética , Melanoma/tratamiento farmacológico , Melanoma/patología , Femenino , Masculino , Persona de Mediana Edad , Adulto , Anciano , Proteínas Proto-Oncogénicas B-raf/genética , GTP Fosfohidrolasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas c-raf/genética , Anciano de 80 o más Años , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in sarcomeric genes. However, a subset of cases is attributed to genetic disorders unrelated to sarcomeric genes, such as Noonan syndrome (NS) and other RASopathies. In this study, we present a family with a history of sudden cardiac death (SCD) and focus on two adults with syndromic left ventricular hypertrophy (LVH). METHODS: Clinical evaluations, including echocardiography, were conducted to assess cardiac manifestations. Whole-exome sequencing was performed to identify potential genetic variants underlying syndromic LVH in the study participants. RESULTS: Whole-exome sequencing revealed a missense variant in the RAF1 gene, c.782C>T (p.Pro261Leu). This variant confirmed the diagnosis of NS in the affected individuals. CONCLUSION: The findings of this study underscore the importance of family history investigation and genetic testing in diagnosing syndromic LVH. By identifying the underlying genetic cause, clinicians can better understand the etiology of RAS-HCM and its association with SCD in young adults.
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Cardiomiopatía Hipertrófica , Síndrome de Noonan , Humanos , Adulto Joven , Cardiomiopatía Hipertrófica/diagnóstico , Cardiomiopatía Hipertrófica/genética , China , Muerte Súbita Cardíaca/etiología , Mutación , Síndrome de Noonan/diagnóstico , Síndrome de Noonan/genética , Proteínas Proto-Oncogénicas c-raf/genéticaRESUMEN
The RAS/mitogen-activated protein kinase (MAPK) signaling cascade is commonly dysregulated in human malignancies by processes driven by RAS or RAF oncogenes. Among the members of the RAF kinase family, CRAF plays an important role in the RAS-MAPK signaling pathway, as well as in the progression of cancer. Recent research has provided evidence implicating the role of CRAF in the physiological regulation and the resistance to BRAF inhibitors through MAPK-dependent and MAPK-independent mechanisms. Nevertheless, the effectiveness of solely targeting CRAF kinase activity remains controversial. Moreover, the kinase-independent function of CRAF may be essential for lung cancers with KRAS mutations. It is imperative to develop strategies to enhance efficacy and minimize toxicity in tumors driven by RAS or RAF oncogenes. The review investigates CRAF alterations observed in cancers and unravels the distinct roles of CRAF in cancers propelled by diverse oncogenes. This review also seeks to summarize CRAF-interacting proteins and delineate CRAF's regulation across various cancer hallmarks. Additionally, we discuss recent advances in pan-RAF inhibitors and their combination with other therapeutic approaches to improve treatment outcomes and minimize adverse effects in patients with RAF/RAS-mutant tumors. By providing a comprehensive understanding of the multifaceted role of CRAF in cancers and highlighting the latest developments in RAF inhibitor therapies, we endeavor to identify synergistic targets and elucidate resistance pathways, setting the stage for more robust and safer combination strategies for cancer treatment.
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Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas B-raf , Humanos , Línea Celular Tumoral , Transducción de Señal , Fosforilación , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
BACKGROUND: Women are at greater risk of developing non-small cell lung cancer (NSCLC), yet the underlying causes remain unclear. METHODS: We performed whole genome scans in lung tumours of cRaf transgenic mice and identified miRNA, transcription factor and hormone receptor dependent gene regulations. We confirmed hormone receptors by immunohistochemistry and constructed regulatory gene networks by considering experimentally validated miRNA-gene and transcription factor-miRNA/gene targets. Bioinformatics, genomic foot-printing and gene enrichment analysis established sex-specific circuits of lung tumour growth. Translational research involved a large cohort of NSCLC patients. We evaluated commonalities in sex-specific NSCLC gene regulations between mice and humans and determined their prognostic value in Kaplan-Meier survival statistics and COX proportional hazard regression analysis. FINDINGS: Overexpression of the cRaf kinase elicited an extraordinary 8-fold increase in tumour growth among females, and nearly 70% of the 112 differentially expressed genes (DEGs) were female specific. We identified oncogenes, oncomirs, tumour suppressors, cell cycle regulators and MAPK/EGFR signalling molecules, which prompted sex-based differences in NSCLC, and we deciphered a regulatory gene-network, which protected males from accelerated tumour growth. Strikingly, 41% of DEGs are targets of hormone receptors, and the majority (85%) are oestrogen receptor (ER) dependent. We confirmed the role of ER in a large cohort of NSCLC patients and validated 40% of DEGs induced by cRaf in clinical tumour samples. INTERPRETATION: We report the molecular wiring that prompted sex disparities in tumour growth. This allowed us to propose the development of molecular targeted therapies by jointly blocking ER, CDK1 and arginase 2 in NSCLC. FUNDING: We gratefully acknowledge the financial support of the Lower Saxony Ministry of Culture and Sciences and Volkswagen Foundation, Germany to JB (25A.5-7251-99-3/00) and of the Chinese Scholarship Council to SZ (202008080022). This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the "Open Access Publikationskosten" program.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , MicroARNs , Animales , Femenino , Humanos , Masculino , Ratones , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptores de Estrógenos/metabolismoRESUMEN
Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.
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Cardiomiopatía Hipertrófica , Síndrome de Noonan , Proteínas Proto-Oncogénicas c-raf , Humanos , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Mutación de Línea Germinal , Miocitos Cardíacos/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/complicaciones , Síndrome de Noonan/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-raf/genéticaRESUMEN
Somatic and germline gain-of-function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor-prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss-of-function of the proto-oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress-induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.
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Síndrome de Noonan , Proteínas Tirosina Quinasas Receptoras , Humanos , Recién Nacido , Desarrollo Embrionario/genética , Corazón , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Xenopus laevis/genéticaRESUMEN
RAF kinases play major roles in cancer. BRAFV600E mutants drive ~6% of human cancers. Potent kinase inhibitors exist but show variable effects in different cancer types, sometimes even inducing paradoxical RAF kinase activation. Both paradoxical activation and drug resistance are frequently due to enhanced dimerization between RAF1 and BRAF, which maintains or restores the activity of the downstream MEK-ERK pathway. Here, using quantitative proteomics we mapped the interactomes of RAF1 monomers, RAF1-BRAF and RAF1-BRAFV600E dimers identifying and quantifying >1,000 proteins. In addition, we examined the effects of vemurafenib and sorafenib, two different types of clinically used RAF inhibitors. Using regression analysis to compare different conditions we found a large overlapping core interactome but also distinct condition specific differences. Given that RAF proteins have kinase independent functions such dynamic interactome changes could contribute to their functional diversification. Analysing this dataset may provide a deeper understanding of RAF signalling and mechanisms of resistance to RAF inhibitors.
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Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas c-raf , Humanos , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal , Vemurafenib , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genética , ProteomaRESUMEN
The germline mutations in the C-terminus of CRAF kinase, particularly L603, and S612T/L613V, are associated with congenital heart disorders, for example, dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). The experimental data suggest that genetic alternation at position 603 impairs, while those at positions 612/613 enhance the CRAF kinase activity. However, the underlying mechanistic details by which these mutations increase or decrease kinase activity remain elusive. Therefore, we applied molecular dynamic simulation to investigate the impacts of these point mutations on the conformation of the CRAF kinase domain. The results revealed that the substitution of Leucine 603 for proline transits the kinase domain to a state that exhibits the molecular hallmarks of an inactive kinase, for example, a closed activation loop, 'αC-helix out' conformation and a distorted regulatory hydrophobic spine. However, two HCM-associated variants (S612T and L613V) show features of an active conformation, such as an open activation loop conformation, 'αC-helix in', the assembly of the hydrophobic spine, and more surface-exposed catalytic residues of phosphoryl transfer reaction. Overall, our study provides a mechanistic basis for the contradictory effects of the CRAF variants associated with HCM and DCM.
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Cardiomiopatía Hipertrófica , Proteínas Proto-Oncogénicas c-raf , Humanos , Cardiomiopatías/genética , Cardiomiopatía Hipertrófica/genética , Mutación , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genéticaRESUMEN
The protein rapidly accelerated fibrosarcoma (RAF) is a kinase downstream of the membrane protein RAS in the cellular signal transduction system. In the structure of RAF, the N- and C-terminus domains are connected with a flexible linker. The open/close dynamics and dimerization of RAF are thought to regulate its activity, although the details of these conformations are unknown, especially in live cells. In this work, we used alternating laser excitation to measure cytosolic CRAF in live HeLa cells and obtained single-molecule Förster resonance energy transfer (smFRET) distributions of the structural states. We compared the results for wild-type (WT)-CRAF before and after epidermal growth factor (EGF) stimulation, with mutations of the 14-3-3 binding sites and cysteine-rich domain, and an N-terminus truncation. The smFRET distributions of full-length CRAFs were analyzed by global fitting with three beta distributions. Our results suggested that a 14-3-3 dimer bound to two sites on a single CRAF molecule and induced the formation of the autoinhibitory closed conformation. There were two closed conformations, which the majority of WT-CRAF adopted. These two conformations showed different responsiveness to EGF stimulation.
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Proteínas 14-3-3 , Proteínas Proto-Oncogénicas c-raf , Humanos , Cisteína/química , Factor de Crecimiento Epidérmico/metabolismo , Células HeLa , Dominios Proteicos , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genética , Unión Proteica , Secuencias de Aminoácidos , Proteínas 14-3-3/químicaRESUMEN
Phenotype analysis of the Noonan syndrome (NS) related to RAF1 mutations demonstrates that a high proportion of cases exhibit severe lymphatic dysplasia and congenital heart disease, especially hypertrophic cardiomyopathy. Because of the difficulty of fetal phenotypic assessment, the percentage of cases with multisystemic prenatal presentation as well as the phenotypic variability may be underestimated. We describe a 35 weeks male preterm infant presenting with de novo missense mutation NM_002880.4(RAF1):c.770C>T (p.Ser257Leu), whose death occurred following birth. Antenatal ultrasound showed polyhydramnios, severe ascites, and tongue protrusion. Autopsy revealed multiple congenital anomalies including intrauterine growth restriction, hydrops fetalis, characteristic facial dysmorphia, short and webbed neck, hypertrichosis, severe lungs hypoplasia, thymic hyperplasia, hepato-splenomegaly, bilateral mild uretero-hydronephrosis, and mild pontocerebellar hypoplasia. Histology revealed increased hepatic hematopoiesis and iron deposits. This report confirms that NS may be associated with multisystem involvement and provides further evidence for the wide phenotypic variability associated with RAF1 variants.
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Cardiopatías Congénitas , Síndrome de Noonan , Recién Nacido , Humanos , Masculino , Femenino , Embarazo , Proteínas Proto-Oncogénicas c-raf/genética , Recien Nacido Prematuro , Cardiopatías Congénitas/diagnóstico , Cardiopatías Congénitas/genética , Síndrome de Noonan/genética , Hidropesía Fetal/diagnóstico , Hidropesía Fetal/genética , FenotipoRESUMEN
BACKGROUND & AIMS: The contribution of abnormal metabolic targets to hepatocellular carcinoma (HCC) progression and the associated regulatory mechanisms are attractive research areas. High-density lipoprotein binding protein (HDLBP) is an important transporter that protects cells from excessive cholesterol accumulation, but few studies have identified a role for HDLBP in HCC progression. METHODS: HDLBP expression was determined in HCC tissues and published datasets. The biological roles of HDLBP in vitro and in vivo were examined by performing a series of functional experiments. RESULTS: An integrated analysis confirmed that HDLBP expression was significantly elevated in HCC compared with noncancerous liver tissues. The knockdown or overexpression of HDLBP substantially inhibited or enhanced, respectively, HCC proliferation and sorafenib resistance. Subsequently, a mass spectrometry screen identified RAF1 as a potential downstream target of HDLBP. Mechanistically, when RAF1 was stabilized by HDLBP, MEKK1 continuously induced RAF1Ser259-dependent MAPK signaling. Meanwhile, HDLBP interacted with RAF1 by competing with the TRIM71 E3 ligase and inhibited RAF1 degradation through the ubiquitin-proteasome pathway. CONCLUSIONS: Our study reveals that HDLBP is an important mediator that stabilizes the RAF1 protein and maintains its activity, leading to HCC progression and sorafenib resistance. Thus, HDLBP might represent a potential biomarker and future therapeutic target for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Proto-Oncogénicas c-raf , Proteínas de Motivos Tripartitos , Humanos , Biomarcadores , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Proliferación Celular , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Sorafenib/farmacología , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The incidence of colorectal cancer (CRC) is rising worldwide. Here, we identified SCNN1B as an outlier down-regulated in CRC and it functions as a tumor suppressor. SCNN1B mRNA and protein expression were down-regulated in primary CRC and CRC cells. In a tissue microarray cohort (N = 153), SCNN1B protein was an independent prognostic factor for favorable outcomes in CRC. Ectopic expression of SCNN1B in CRC cell lines suppressed cell proliferation, induced apoptosis, and cell cycle arrest, and suppressed cell migration in vitro. Xenograft models validated tumor suppressive function of SCNN1B in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) showed that SCNN1B correlates with KRAS signaling. Consistently, MAPK qPCR and kinase arrays revealed that SCNN1B suppressed MAPK signaling. In particular, SCNN1B overexpression suppressed p-MEK/p-ERK expression and SRE-mediated transcription activities, confirming blockade of Ras-Raf-MEK-ERK cascade. Mechanistically, SCNN1B did not affect KRAS activation, instead impairing activation of c-Raf by inducing its inhibitory phosphorylation and targeting active c-Raf for degradation. The ectopic expression of c-Raf fully rescued cell proliferation and colony formation in SCNN1B-overexpressing CRC cells, confirming c-Raf as the principal molecular target of SCNN1B. In summary, we identified SCNN1B as a tumor suppressor by functioning as a c-Raf antagonist, which in turn suppressed oncogenic MEK-ERK signaling.
Asunto(s)
Neoplasias Colorrectales , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Canales Epiteliales de Sodio/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Canales de Sodio/metabolismoRESUMEN
BACKGROUND: Noonan syndrome is an autosomal dominant genetic disorder that can occur in men and women and has a sporadic or family history. NS can lead to abnormal bleeding, but cerebral haemorrhage is rare. This is the first case of cerebral haemorrhage with a RAF1 gene mutation that originated in the neonatal period. CASE PRESENTATION: This case presents a newborn with a RAF1 gene mutation resulting in NS complicated with an abnormality of chromosome 46, X, del (Y) (q12). In the course of treatment, the baby's breathing suddenly increased. After an MRI examination of the skull, haemorrhaging was found in multiple parts of the brain. CONCLUSIONS: After symptomatic treatment, the baby recovered well, but the main cause of cerebral haemorrhage was not found.
Asunto(s)
Síndrome de Noonan , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Mutación/genética , Síndrome de Noonan/complicaciones , Síndrome de Noonan/genética , Proteínas Proto-Oncogénicas c-raf/genéticaRESUMEN
We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1âS cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.
Asunto(s)
Ciclina D1 , Proteína Quinasa C-alfa , Ciclina D1/genética , Ciclina D1/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
We recently reported an RAF rearrangement without NRAS or BRAF mutations in lesions from Giant Congenital Melanocytic Nevi (CMN). The new gene fusion involves the 5'-end of the promoter-containing N terminus of the SOX5 gene fused to exons 7-16 of the 3'-end of RAF1 gene leading to a SOX5-RAF1 fusion transcript which loses the auto-inhibitory CR1 domain but retains the complete in-frame coding sequence for the C-Terminal kinase domain of the RAF1. Stable expression of SOX5-RAF1 fusion induced growth factor-independent cell growth in murine hematopoietic Ba/F3 cells and melan-a immortalized melanocytes. Besides, it led to the transformation of both Ba/F3 and NIH 3T3 cells as revealed by colony formation assays. Furthermore, its expression results in MAPK activation assessed by increased levels of p-ERK protein in the cytosol of transduced cells. Treatment with Sorafenib and UO126 inhibited proliferation of Ba/F3-SOX5-RAF1 cells in the absence of IL3 but not the PLX 4720, a specific inhibitor of BRAF. Moreover, the tumorigenic and metastatic capacities of SOX5-RAF1 were assessed in vivo. These results indicate that SOX5-RAF1, a driver event for CMN development, has oncogenic capacity. Thus, sequencing of CMN transcriptomes may lead to the identification of this druggable fusion and interfere with the progression toward melanoma.