RESUMEN
Porcine deltacoronavirus (PDCoV), an enteropathogenic coronavirus, causes severe watery diarrhoea, dehydration and high mortality in piglets, which has the potential for cross-species transmission in recent years. Growth factor receptor-bound protein 2 (Grb2) is a bridging protein that can couple cell surface receptors with intracellular signal transduction events. Here, we investigated the reciprocal regulation between Grb2 and PDCoV. It is found that Grb2 regulates PDCoV infection and promotes IFN-ß production through activating Raf/MEK/ERK/STAT3 pathway signalling in PDCoV-infected swine testis cells to suppress viral replication. PDCoV N is capable of interacting with Grb2. The proline-rich motifs in the N- or C-terminal region of PDCoV N were critical for the interaction between PDCoV-N and Grb2. Except for Deltacoronavirus PDCoV N, the Alphacoronavirus PEDV N protein could interact with Grb2 and affect the regulation of PEDV replication, while the N protein of Betacoronavirus PHEV and Gammacoronavirus AIBV could not interact with Grb2. PDCoV N promotes Grb2 degradation by K48- and K63-linked ubiquitin-proteasome pathways. Overexpression of PDCoV N impaired the Grb2-mediated activated effect on the Raf/MEK/ERK/STAT3 signal pathway. Thus, our study reveals a novel mechanism of how host protein Grb2 protein regulates viral replication and how PDCoV N escaped natural immunity by interacting with Grb2.
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Proteína Adaptadora GRB2 , Proteínas de la Nucleocápside , Replicación Viral , Animales , Porcinos , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/metabolismo , Deltacoronavirus/metabolismo , Deltacoronavirus/genética , Sistema de Señalización de MAP Quinasas , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/metabolismo , Humanos , Transducción de Señal , Línea Celular , Quinasas raf/metabolismo , Quinasas raf/genética , Células HEK293RESUMEN
In this issue, Ryan and colleagues describe the preclinical development of a pan-RAF:MEK molecular glue with superior efficacy, brain penetrance, and tolerability in xenograft models of Ras/Raf/MAPK pathway-driven tumors. See related article by Ryan et al., p. 1190 (1).
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Inhibidores de Proteínas Quinasas , Humanos , Animales , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Quinasas raf/metabolismo , Quinasas raf/genética , Ratones , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
BACKGROUND: Mitogen-activated protein kinase (MAPK)signaling-mediated smoking-associated pulmonary vascular remodeling (PVR) plays an important role in the pathogenesis of group 3 pulmonary hypertension (PH). And G protein pathway suppressor 2 (GPS2) could suppress G-protein signaling such as Ras and MAPK, but its role in cigarette smoking -induced PVR (CS-PVR) is unclear. METHODS: An in vivo model of smoke-exposed rats was constructed to assess the role of GPS2 in smoking-induced PH and PVR. In vitro, the effects of GPS2 overexpression and silencing on the function of human pulmonary arterial smooth cells (HPASMCs) and the underlying mechanisms were explored. RESULTS: GPS2 expression was downregulated in rat pulmonary arteries (PAs) and HPASMCs after CS exposure. More importantly, CS-exposed rats with GPS2 overexpression had lower right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness (WT%) than those without. And enhanced proliferation and migration of HPASMCs induced by cigarette smoking extract (CSE) can be evidently inhibited by overexpressed GPS2. Besides, GPS2siRNA significantly enhanced the proliferation, and migration of HPASMCs as well as activated Ras and Raf/ERK signaling, while these effects were inhibited by zoledronic acid (ZOL). In addition, GPS2 promoter methylation level in rat PAs and HPASMCs was increased after CS exposure, and 5-aza-2-deoxycytidine (5-aza) inhibited CSE-induced GPS2 hypermethylation and downregulation in vitro. CONCLUSIONS: GPS2 overexpression could improve the CS-PVR, suggesting that GPS2 might serve as a novel therapeutic target for PH-COPD in the future.
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Fumar Cigarrillos , Sistema de Señalización de MAP Quinasas , Ratas Sprague-Dawley , Remodelación Vascular , Animales , Remodelación Vascular/efectos de los fármacos , Remodelación Vascular/fisiología , Ratas , Masculino , Humanos , Fumar Cigarrillos/efectos adversos , Sistema de Señalización de MAP Quinasas/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Cultivadas , Proteínas ras/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Quinasas raf/metabolismo , Quinasas raf/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/inducido químicamente , Quinasas MAP Reguladas por Señal Extracelular/metabolismoRESUMEN
Lung cancer, despite recent advancements in survival rates, represents a significant global health burden. Non-small cell lung cancer (NSCLC), the most prevalent type, is driven largely by activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and receptor tyrosine kinases (RTKs), and less in v-RAF murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein-kinase kinase (MEK), all key components of the RTK-RAS-mitogen-activated protein kinase (MAPK) pathway. Learning from melanoma, the identification of BRAFV600E substitution in NSCLC provided the rationale for the investigation of RAF and MEK inhibition as a therapeutic strategy. The regulatory approval of two RAF-MEK inhibitor combinations, dabrafenib-trametinib, in 2017, and encorafenib-binimetinib, in 2023, signifies a breakthrough for the management of BRAFV600E-mutant NSCLC patients. However, the almost universal emergence of acquired resistance limits their clinical benefit. New RAF and MEK inhibitors, with distinct biochemical characteristics, are in preclinical and clinical development. In this review, we aim to provide valuable insights into the current state of RAF and MEK inhibition in the management of NSCLC, fostering a deeper understanding of the potential impact on patient outcomes.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Quinasas de Proteína Quinasa Activadas por Mitógenos , Inhibidores de Proteínas Quinasas , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Animales , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismo , Quinasas raf/genética , MutaciónRESUMEN
OBJECTIVES: Uterine carcinosarcomas (UCS) are rare, biologically aggressive tumors. Since UCS may harbor mutations in RAS/MAPK pathway genes we evaluated the preclinical in vitro and in vivo efficacy of the RAF/MEK clamp avutometinib in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718 against multiple primary UCS cell lines and xenografts. METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic landscape of 5 primary UCS cell lines. The in vitro activity of avutometinib ± FAK inhibitor was evaluated using cell viability and cell cycle assays against primary UCS cell lines. Mechanistic studies were performed using western blot assays while in vivo experiments were completed in UCS tumor bearing mice treated with avutometinib ± FAK inhibitor by oral gavage. RESULTS: WES results demonstrated multiple UCS cell lines harbor genetic alterations including KRAS, PTK2, BRAF, MAP2K, and MAP2K1, potentially sensitizing to FAK and RAF/MEK inhibition. Four out of five of the UCS cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition when used alone or in combination. By western blot assays, exposure of UCS cell lines to the combination of defactinib/avutometinib demonstrated decreased phosphorylated (p)-FAK as well as decreased p-ERK. In vivo, the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition and longer survival compared to single agent treatment and controls starting at day 10 (p < 0.002) in UCS xenografts. CONCLUSION: The combination of avutometinib and defactinib demonstrates promising in vitro and in vivo anti-tumor activity against primary UCS cell lines and xenografts.
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Carcinosarcoma , Neoplasias Uterinas , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Humanos , Animales , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/patología , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Línea Celular Tumoral , Ratones , Carcinosarcoma/tratamiento farmacológico , Carcinosarcoma/patología , Carcinosarcoma/genética , Carcinosarcoma/metabolismo , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Indoles/farmacología , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismo , Quinasas raf/genética , Secuenciación del Exoma , Ratones Desnudos , Benzamidas , Pirazinas , SulfonamidasRESUMEN
ABSTRACT: Multiple myeloma is characterized by a huge heterogeneity at the molecular level. The RAS/RAF pathway is the most frequently mutated, in â¼50% of the patients. However, these mutations are frequently subclonal, suggesting a secondary event. Because these genes are part of our routine next-generation sequencing panel, we analyzed >10 000 patients with different plasma cell disorders to describe the RAS/RAF landscape. In this large cohort of patients, almost 61% of the patients presented a RAS/RAF mutation at diagnosis or relapse, but much lower frequencies occurred in presymptomatic cases. Of note, the mutations were different from that observed in solid tumors (higher proportions of Q61 mutations). In 29 patients with 2 different mutations, we were able to perform single-cell sequencing, showing that in most cases, mutations occurred in different subclones, suggesting an ongoing mutational process. These findings suggest that the RAS/RAF pathway is not an attractive target, both on therapeutic and residual disease assessment points of view.
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Mieloma Múltiple , Mutación , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Proteínas ras/genética , Proteínas ras/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.
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Sirtuinas , Humanos , Células HEK293 , Sirtuinas/genética , Sirtuinas/metabolismo , Transducción de Señal , Mitocondrias/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismoRESUMEN
Mutations in the RAS-RAF-MEK-ERK pathway are frequent alterations in cancer and RASopathies, and while RAS oncogene activation alone affects 19% of all patients and accounts for approximately 3.4 million new cases every year, less frequent alterations in the cascade's downstream effectors are also involved in cancer etiology. RAS proteins initiate the signaling cascade by promoting the dimerization of RAF kinases, which can act as oncoproteins as well: BRAFV600E is the most common oncogenic driver, mutated in the 8% of all malignancies. Research in this field led to the development of drugs that target the BRAFV600-like mutations (Class I), which are now utilized in clinics, but cause paradoxical activation of the pathway and resistance development. Furthermore, they are ineffective against non-BRAFV600E malignancies that dimerize and could be either RTK/RAS independent or dependent (Class II and III, respectively), which are still lacking an effective treatment. This review discusses the recent advances in anti-RAF therapies, including paradox breakers, dimer-inhibitors, immunotherapies, and other novel approaches, critically evaluating their efficacy in overcoming the therapeutic limitations, and their putative role in blocking the RAS pathway.
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Neoplasias , Quinasas raf , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Quinasas raf/metabolismo , Quinasas raf/antagonistas & inhibidores , Quinasas raf/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Animales , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Mutación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Terapia Molecular DirigidaRESUMEN
RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).
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Transducción de Señal , Quinasas raf , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Proteínas ras/química , Quinasas raf/metabolismo , Quinasas raf/genética , Animales , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genéticaRESUMEN
BACKGROUND: The prognosis of malignant primary high-grade brain tumors, predominantly glioblastomas, is poor despite intensive multimodality treatment options. In more than 50% of patients with glioblastomas, potentially targetable mutations are present, including rearrangements, altered splicing, and/or focal amplifications of epidermal growth factor receptor (EGFR) by signaling through the RAF/RAS pathway. We studied whether treatment with the clinically available anti-EGFR monoclonal antibody panitumumab provides clinical benefit for patients with RAF/RAS-wild-type (wt) glioblastomas in the Drug Rediscovery Protocol (DRUP). METHODS: Patients with progression of treatment refractory RAF/RASwt glioblastoma were included for treatment with panitumumab in DRUP when measurable according to RANO criteria. The primary endpoints of this study are clinical benefit (CB: defined as confirmed objective response [OR] or stable disease [SD] ≥ 16 weeks) and safety. Patients were enrolled using a Simon-like 2-stage model, with 8 patients in stage 1 and up to 24 patients in stage 2 if at least 1 in 8 patients had CB in stage 1. RESULTS: Between 03-2018 and 02-2022, 24 evaluable patients were treated. CB was observed in 5 patients (21%), including 2 patients with partial response (8.3%) and 3 patients with SD ≥ 16 weeks (12.5%). After median follow-up of 15 months, median progression-free survival and overall survival were 1.7 months (95% CI 1.6-2.1 months) and 4.5 months (95% CI 2.9-8.6 months), respectively. No unexpected toxicities were observed. CONCLUSIONS: Panitumumab treatment provides limited CB in patients with recurrent RAF/RASwt glioblastoma precluding further development of this therapeutic strategy.
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Glioblastoma , Panitumumab , Humanos , Panitumumab/uso terapéutico , Panitumumab/efectos adversos , Panitumumab/farmacología , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/mortalidad , Masculino , Persona de Mediana Edad , Anciano , Adulto , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Antineoplásicos Inmunológicos/uso terapéutico , Antineoplásicos Inmunológicos/efectos adversos , Proteínas ras/genética , Quinasas raf/genética , Quinasas raf/antagonistas & inhibidoresRESUMEN
Metastatic dissemination of solid tumors, a leading cause of cancer-related mortality, underscores the urgent need for enhanced insights into the molecular and cellular mechanisms underlying metastasis, chemoresistance, and the mechanistic backgrounds of individuals whose cancers are prone to migration. The most prevalent signaling cascade governed by multi-kinase inhibitors is the mitogen-activated protein kinase (MAPK) pathway, encompassing the RAS-RAF-MAPK kinase (MEK)-extracellular signal-related kinase (ERK) pathway. RAF kinase is a primary mediator of the MAPK pathway, responsible for the sequential activation of downstream targets, such as MEK and the transcription factor ERK, which control numerous cellular and physiological processes, including organism development, cell cycle control, cell proliferation and differentiation, cell survival, and death. Defects in this signaling cascade are associated with diseases such as cancer. RAF inhibitors (RAFi) combined with MEK blockers represent an FDA-approved therapeutic strategy for numerous RAF-mutant cancers, including melanoma, non-small cell lung carcinoma, and thyroid cancer. However, the development of therapy resistance by cancer cells remains an important barrier. Autophagy, an intracellular lysosome-dependent catabolic recycling process, plays a critical role in the development of RAFi resistance in cancer. Thus, targeting RAF and autophagy could be novel treatment strategies for RAF-mutant cancers. In this review, we delve deeper into the mechanistic insights surrounding RAF kinase signaling in tumorigenesis and RAFi-resistance. Furthermore, we explore and discuss the ongoing development of next-generation RAF inhibitors with enhanced therapeutic profiles. Additionally, this review sheds light on the functional interplay between RAF-targeted therapies and autophagy in cancer.
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Neoplasias Pulmonares , Melanoma , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.
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Quinasas raf , Proteínas ras , Humanos , Sitio Alostérico , Hidrólisis , Quinasas raf/química , Quinasas raf/genética , Quinasas raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismoRESUMEN
Glyphosate is an herbicide commonly used worldwide, and its substantial use causes widespread pollution with runoff. However, research on glyphosate toxicity has mostly remained at the embryonic level and existing studies are limited. In the present study, we investigated whether glyphosate can induce autophagy in hepatic L8824 cells by regulating energy metabolism and rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling by activating nitric oxide (NO). First, we selected 0, 50, 200, and 500 µg/mL as the challenge doses, according to the inhibitory concentration of 50% (IC50) of glyphosate. The results showed that glyphosate exposure increased the enzyme activity of inducible nitric oxide synthase (iNOS), which in turn increased the NO content. The activity and expression of enzymes related to energy metabolism, such as hexokinase (HK)1, HK2, phosphofructokinase (PFK), phosphokinase (PK), succinate dehydrogenase (SDH), and nicotinamide adenine dinucleotide with hydrogen (NADH), were inhibited, and the RAS/RAF/MEK/ERK signaling pathway was activated. This led to the negative expression of mammalian target of rapamycin (mTOR) and P62 in hepatic L8824 cells and the activation of the autophagy marker genes microtubule-associated proteins light chain 3 (LC3) and Beclin1 to induce autophagy. The above results were dependent on glyphosate concentration. To verify whether autophagy can be excited by the RAS/RAF/MEK/ERK signaling pathway, we treated L8824 cells with the ERK inhibitor U0126 and found that the autophagy gene LC3 was reduced due to the inhibition of ERK, thus demonstrating the reliability of the results. In conclusion, our results demonstrate that glyphosate can induce autophagy in hepatic L8824 cells by activating NO, thus regulating energy metabolism and the RAS/RAF/MEK/ERK signaling pathway.
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Fibrosarcoma , Quinasas Quinasa Quinasa PAM , Animales , Óxido Nítrico , Reproducibilidad de los Resultados , Quinasas raf/genética , Transducción de Señal , Quinasas MAP Reguladas por Señal Extracelular , Línea Celular , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Metabolismo Energético , Autofagia , Sistema de Señalización de MAP Quinasas , Mamíferos/metabolismo , GlifosatoRESUMEN
One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.
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Quinasas raf , Proteínas ras , Dimerización , Consenso , Proteínas ras/genética , Proteínas ras/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Lípidos , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.
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Síndrome de Noonan , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Fosfoserina/metabolismo , Proteína Fosfatasa 1 , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
BACKGROUND: Aldehyde dehydrogenase 6 family member A1 (ALDH6A1) is associated with multiple diseases, but its pathogenesis in colon cancer (CC) is ambiguous and needs further study so that this research explores the function of ALDH6A1 in CC. METHODS: The level of ALDH6A1 in colon adenocarcinoma (COAD), CC tissues, and cells was measured by starBase v2.0, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. Post transfection with overexpressed (oe)-ALDH6A1, cell biological behaviors, as well as apoptosis-, matrix metalloproteinase (MMP)-, and rat sarcoma virus (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway-related markers were measured by cell function experiments, qRT-PCR, and western blot. Next, the effects of small interfering RNA targeting ALDH6A1 (si-ALDH6A1) and RAS/RAF inhibitor (MCP110) on cell biological behaviors, as well as apoptosis-, MMP-, and RAS/RAF/MEK/ERK pathway-related markers were detected again. RESULTS: ALDH6A1 was low-expressed in COAD, CC tissues, and cells. Oe-ALDH6A1 weakened cell vitality, migration and invasionbut facilitated apoptosis; while it reduced expression levels of Bcl-2, MMP-2, MMP-9 and the RAS/RAF/MEK/ERK pathway-related markers but promoted Bax level. However, the regulation of si-ALDH6A1 on cell biological behaviors and related genes was opposite to that of oe-ALDH6A1. Moreover, MCP110 rescued the regulation of si-ALDH6A1 on cell biological behaviors, expressions of apoptosis- MMP- as well as RAS/RAF/MEK/ERK pathway-related markers. To sum up, ALDH6A1 attenuated CC progression by down-regulating the expressions of RAS/RAF/MEK/ERK pathway-related markers.
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Adenocarcinoma , Neoplasias del Colon , Fibrosarcoma , Línea Celular , Neoplasias del Colon/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
The RAS GTPases are frequently mutated in human cancer, with KRAS being the predominant tumor driver. For many years, it has been known that the structure and function of RAS are integrally linked, as structural changes induced by GTP binding or mutational events determine the ability of RAS to interact with regulators and effectors. Recently, a wealth of information has emerged from structures of specific KRAS mutants and from structures of multiprotein complexes containing RAS and/or RAF, an essential effector of RAS. These structures provide key insights regarding RAS and RAF regulation as well as promising new strategies for therapeutic intervention. SIGNIFICANCE: The RAS GTPases are major drivers of tumorigenesis, and for RAS proteins to exert their full oncogenic potential, they must interact with the RAF kinases to initiate ERK cascade signaling. Although binding to RAS is typically a prerequisite for RAF to become an activated kinase, determining the molecular mechanisms by which this interaction results in RAF activation has been a challenging task. A major advance in understanding this process and RAF regulation has come from recent structural studies of various RAS and RAF multiprotein signaling complexes, revealing new avenues for drug discovery.
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Quinasas raf , Proteínas ras , Humanos , Sistema de Señalización de MAP Quinasas , Oncogenes , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Quinasas raf/genética , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven mouse melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF partner. Interestingly, this interaction is conserved among the RAF protein kinase family since BRAF/MITF and CRAF/MITF complexes were also observed in the cytosol of NRAS-mutated mouse melanoma cells. The interaction occurs through the kinase domain of RAF proteins. Importantly, endogenous BRAF/MITF complexes were also detected in BRAF-mutated human melanoma cells. RAF/MITF complexes modulate MITF nuclear localization by inducing an accumulation of MITF in the cytoplasm, thus negatively controlling its transcriptional activity. Taken together, our study highlights a new level of regulation between two major mediators of melanoma progression, MITF and the MAPK/ERK pathway, which appears more complex than previously anticipated.
Asunto(s)
Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Quinasas raf/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Quinasas raf/genéticaRESUMEN
Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of rhabdomyosarcoma (RMS). Among RMS subtypes, ERMS is associated with a favorable outcome with an overall survival of 70% at 5 years for localized disease. The molecular profile of ERMS is heterogeneous, including mostly point mutations in various genes. Therapeutic strategies have remained relatively consistent irrespective of the molecular abnormalities. In this study, we focus on a homogeneous RAS/RAF mutated ERMS subset and correlate with clinicopathologic findings. Twenty-six cases (16 males and 10 females) were identified from screening 98 ERMS, either by targeted DNA sequencing (MSK-IMPACT) or by Sanger sequencing. Fourteen (54%) cases had NRAS mutations, 6 (23%) had KRAS mutations, 5 (19%) had HRAS mutations, and 1 case (4%) had BRAF mutation. Median age at diagnosis was 8 years (range 1-70) with two-thirds occurring in the children. Tumor sites varied with H&N and GU sites accounting for 62% of cases. RAS isoform hot spot mutations predominated: NRAS p.Q61K (57%), KRAS p.G12D (67%), and HRAS (codons 12, 14, and 61). Additional genetic abnormalities were identified in 85% of the RAS-mutated cases. At last follow-up, 29% of patients died of disease and 23% were alive with disease. The 3-year and 5-year survival rates were 75% and 61% respectively. In conclusion, RAS mutations occur in 27% of ERMS, with NRAS mutations encompassing half of the cases. Overall RAS-mutant RMS does not correlate with age or site, but most tumors show an undifferentiated and spindle cell morphology.
Asunto(s)
Mutación/genética , Rabdomiosarcoma Embrionario , Quinasas raf/genética , Proteínas ras/genética , Adulto , Anciano , Niño , Preescolar , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Rabdomiosarcoma Embrionario/genética , Rabdomiosarcoma Embrionario/mortalidad , Rabdomiosarcoma Embrionario/patología , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/mortalidad , Neoplasias de los Tejidos Blandos/patología , Neoplasias Urogenitales/genética , Neoplasias Urogenitales/mortalidad , Neoplasias Urogenitales/patología , Adulto JovenRESUMEN
Aberrant activity of oncogenic rat sarcoma virus (RAS) protein promotes tumor growth and progression. RAS-driven cancers comprise more than 30% of all human cancers and are refractory to frontline treatment strategies. Since direct targeting of RAS has proven challenging, efforts have been centered on the exploration of inhibitors for RAS downstream effector kinases. Two major RAS downstream signaling pathways, including the Raf/MEK/Erk cascade and the phosphatidylinositol-3-kinase (PI3K) pathway, have become compelling targets for RAS-driven cancer therapy. However, the main drawback in the blockade of a single RAS effector is the multiple levels of crosstalk and compensatory mechanisms between these two pathways that contribute to drug resistance against monotherapies. A growing body of evidence reveals that the sequential or synergistic inhibition of multiple RAS effectors is a more convenient route for the efficacy of cancer therapy. Herein, we revisit the recent developments and discuss the most promising modalities targeting canonical RAS downstream effectors for the treatment of RAS-driven cancers.