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1.
Proc Natl Acad Sci U S A ; 117(39): 24258-24268, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32913056

RESUMO

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.


Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismo , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular
2.
Nature ; 583(7815): 265-270, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581361

RESUMO

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.


Assuntos
Segregação de Cromossomos/genética , Evolução Molecular , Genoma/genética , Neoplasias/genética , Alelos , Animais , Reparo do DNA , Replicação do DNA , Receptores ErbB/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Mutação , Neoplasias/patologia , Seleção Genética , Transdução de Sinais , Troca de Cromátide Irmã , Transcrição Genética , Quinases raf/metabolismo , Proteínas ras/metabolismo
3.
Am J Physiol Endocrinol Metab ; 319(1): E232-E244, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369417

RESUMO

Voltage-gated Ca2+ (CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaV channels. Previous studies have shown that EGF increases Ca2+ currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaV channels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 (CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.


Assuntos
Canais de Cálcio Tipo L/genética , Fator de Crescimento Epidérmico/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Proteínas Elk-1 do Domínio ets/genética , Quinases raf/genética , Proteínas ras/genética , Animais , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Técnicas de Silenciamento de Genes , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo
4.
Nat Commun ; 11(1): 613, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001690

RESUMO

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Pressão Osmótica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Quinases raf/metabolismo , Análise Mutacional de DNA , Mutação/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica
5.
J Neurosci ; 40(14): 2935-2942, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32102921

RESUMO

In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. Here we address this question in the context of sugar-rewarded olfactory long-term memory (LTM). We show that associative training increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf, or MAPK expression in KCs selectively impairs LTM, but not short-term memory. Moreover, we show that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory.SIGNIFICANCE STATEMENT Dopaminergic input to the Kenyon cells (KCs) is pivotal to stabilize memory in Drosophila This process is mediated by dopamine receptors like Dop1R2. Nevertheless, little is known for its underlying molecular mechanism. Here we show that the Raf/MAPK pathway is specifically engaged in appetitive long-term memory in KCs. With combined biochemical and behavioral experiments, we reveal that activation of the Raf/MAPK pathway is regulated through Dop1R2, shedding light on how dopamine modulates intracellular signaling for memory stabilization.


Assuntos
Comportamento Apetitivo/fisiologia , Proteínas de Drosophila/metabolismo , Memória de Longo Prazo/fisiologia , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo , Transdução de Sinais/fisiologia , Animais , Drosophila , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Masculino , Corpos Pedunculados/fisiologia , Quinases raf/metabolismo
6.
Biochem Biophys Res Commun ; 524(4): 970-976, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32059847

RESUMO

Transforming growth factor-ß, a cell secretion factor of the TGF-ß superfamily, is involved in the regulation of cell proliferation, differentiation, cytoskeleton formation, migration, invasion and other biological behaviors. Autophagy and mitophagy play an important role in tumor progression by regulating self-digestion, and degradation and reuse of cells and mitochondria. In this study, changes in autophagy and mitophagy processes in ovarian cancer cells under TGF-ß1 treatment were detected via Western blot and immunofluorescence, as well as the role of fucosylation modification. Changes in mitochondrial membrane potential in response to TGF-ß1 and fucosylation were detected via immunofluorescence. The effects of TGF-ß1 and its fucosylation on autophagic flux were further determined by transient transfection of cells with Ad-mRFP-GFP-LC3 adenovirus. TGF-ß1 clearly promoted autophagy and mitophagy in ovarian cancer cells. TGF-ß1 fucosylation stimulated these regulatory effects on ovarian cancer cells via modulation of PI3K/Akt and Ras-Raf-MEK-ERK pathways through TAK1. Our collective data support the physiological significance of TGF-ß1 and provide a novel direction for targeted therapy for ovarian cancer.


Assuntos
Autofagia , Fucose/metabolismo , Mitofagia , Neoplasias Ovarianas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinases raf/metabolismo , Proteínas ras/metabolismo
7.
Cell Mol Biol Lett ; 25: 1, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31988639

RESUMO

The ubiquitin system, known as a common feature in eukaryotes, participates in multiple cellular processes, such as signal transduction, cell-cycle progression, receptor trafficking and endocytosis, and even the immune response. In lung cancer, evidence has revealed that aberrant events in ubiquitin-mediated processes can cause a variety of pathological outcomes including tumorigenesis and metastasis. Likewise, ubiquitination on the core components contributing to the activity of cell signaling controls bio-signal turnover and cell final destination. Given this, inhibitors targeting the ubiquitin system have been developed for lung cancer therapies and have shown great prospects for clinical application. However, the exact biological effects and physiological role of the drugs used in lung cancer therapies are still not clearly elucidated, which might seriously impede the progress of treatment. In this work, we summarize current research advances in cell signal regulation processes mediated through the ubiquitin system during the development of lung cancer, with the hope of improving the therapeutic effects by means of aiming at efficient targets.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas ras/metabolismo , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Endocitose/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Sistema de Sinalização das MAP Quinases/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina/química , Ubiquitinação/genética , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética
8.
Am J Clin Oncol ; 43(2): 139-145, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31764020

RESUMO

Gynecologic clear cell carcinoma is a rare histology, accounting for ~5% of all ovarian and endometrial cancers in the United States. Compared to other types of gynecologic cancer, they are generally less responsive to standard therapy and have an overall worse prognosis. In addition, mounting evidence suggests that the landscape of genetic and molecular abnormalities observed in these tumors is distinct from other cancers that arise from the same sites of origin. On a molecular level, these tumors characteristically display upregulation of the PI3K-AKT-mTOR and RAS-RAF-MAPK signaling axes, frequent loss of ARID1a, and overexpression of MDM2. Evidence also suggests that these tumors are more likely to express programmed death ligand 1 or demonstrate microsatellite instability than other gynecologic cancers. Despite these important differences, there has been relatively little investigation into histology-specific treatment of clear cell gynecologic cancers, representing an opportunity for new drug development. In this article, we review the unique genetic and molecular features of gynecologic clear cell cancers with an emphasis on potential therapeutic targets. The results of completed studies of treatment for clear cell carcinoma are also presented. We conclude with a discussion of ongoing clinical trials and potential avenues for future study.


Assuntos
Adenocarcinoma de Células Claras/genética , Neoplasias dos Genitais Femininos/genética , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Antineoplásicos Imunológicos/uso terapêutico , Variações do Número de Cópias de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Neoplasias dos Genitais Femininos/tratamento farmacológico , Neoplasias dos Genitais Femininos/metabolismo , Neoplasias dos Genitais Femininos/patologia , Fator 1-beta Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/metabolismo , Humanos , Instabilidade de Microssatélites , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
9.
Artif Cells Nanomed Biotechnol ; 48(1): 37-45, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31852247

RESUMO

Background: Prolyl hydroxylase domain proteins (PHD2) is an oxygen sensor that is able to induce hypoxia-inducible factor-α (HIF-α) degradation under normoxic condition. The present paper designed to reveal the function of PHD2 in hepatocellular carcinoma (HCC) cells proliferation, migration and invasion.Methods: qRT-PCR and Western blot were carried out to see the expression of PHD2 in HCC tissues and cell lines. PHD2 expression in Huh7 and HepG3B cells was overexpressed or suppressed by transfection and then the changes of cell proliferation, migration and invasion were detected by CCK-8 assay, transwell assay and Western blot.Results: PHD2 was highly expressed in HCC tissues and cell lines (Huh7, Hep3B, SK-HEP-1, HCCLM3 and MHCC97) as relative to para-cancerous non-tumour tissues and a normal hepatocyte line MIHA. PHD2 overexpression promoted Huh7 and Hep3B cells viability, migration and invasion. Meanwhile, CyclinD1, c-Myc, MMP-2, MMP-9 and Vimentin were up-regulated, while p53 was down-regulated by PHD2 overexpression. PHD2 silence led to a contrary impact. Further, PHD2 overexpression up-regulated Ras and Raf expression and induced phosphorylation of MEK, ERK, JAK1 and STAT3.Conclusion: PHD2 exhibited pro-tumour functions in HCC cells. PHD2 promoted HCC possibly through Ras/Raf/MEK/ERK and JAK1/STAT3 pathways.HighlightsPHD2 is highly expressed in HCC tissue and cell lines;PHD2 promotes the proliferation of Huh7 and HepG3B cells;PHD2 enhances Huh7 and HepG3B cells migration and invasion;PHD2 activates Ras/Raf/MEK/ERK and JAK1/STAT3 signalling.


Assuntos
Carcinoma Hepatocelular/patologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Neoplasias Hepáticas/patologia , Oncogenes/genética , Transdução de Sinais , Movimento Celular , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 1/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fator de Transcrição STAT3/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo
10.
BMC Cancer ; 19(1): 1160, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783811

RESUMO

BACKGROUND: Syndecan-1 (SDC-1) is a crucial membrane proteoglycan, which is confirmed to participate in several tumor cell biological processes. However, the biological significance of SDC-1 in colorectal carcinoma is not yet clear. An objective of this study was to investigate the role of SDC-1 in colorectal carcinoma cells. METHODS: Expression of SDC-1 in colorectal carcinoma tissues was evaluated by Reverse transcription-quantitative real-time PCR (RT-qPCR) and western blot. After transfection with pcDNA3.1 or pc-SDC-1, the transfection efficiency was measured. Next, SW480, SW620 and LOVO cell viability, apoptosis, migration and adhesion were assessed to explore the effects of exogenous overexpressed SDC-1 on colorectal carcinoma. In addition, the influences of aberrant expressed SDC-1 in Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) and rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways were detected by western blot analysis. RESULTS: SDC-1 mRNA and protein levels were down-regulated in human colorectal carcinoma tissues. SDC-1 overexpression inhibited cell proliferation via suppressing CyclinD1 and c-Myc expression, meanwhile stimulated cell apoptosis via increasing the expression levels of B-cell lymphoma-2-associated x (Bax) and Cleaved-Caspase-3. Additionally, SDC-1 overexpression restrained cell migration via inhibiting the protein expression of matrix metallopeptidase 9 (MMP-9), and elicited cell adhesion through increasing intercellular cell adhesion molecule-1 (ICAM-1). Furthermore, SDC-1 overexpression suppressed JAK1/STAT3 and Ras/Raf/MEK/ERK-related protein levels. CONCLUSIONS: In general, the evidence from this study suggested that SDC-1 suppressed cell growth, migration through blocking JAK1/STAT3 and Ras/Raf/MEK/ERK pathways in human colorectal carcinoma cells.


Assuntos
Neoplasias Colorretais/patologia , Janus Quinase 1/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sindecana-1/metabolismo , Quinases raf/metabolismo , Apoptose , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , Sindecana-1/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
11.
BMC Cancer ; 19(1): 1254, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31883527

RESUMO

BACKGROUND: Bag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer. METHODS: Using immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry. RESULTS: We observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad's re-localization to the nucleus, and inhibits apoptosis in favor of cell survival. CONCLUSIONS: Overall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.


Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/genética , Regulação para Cima , Proteína de Morte Celular Associada a bcl/química , Proteína de Morte Celular Associada a bcl/fisiologia , Quinases raf/metabolismo
12.
BMC Bioinformatics ; 20(1): 507, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638911

RESUMO

BACKGROUND: Human tumor is a complex tissue with multiple heterogeneous hypoxic regions and significant cell-to-cell variability. Due to the complexity of the disease, the explanation of why anticancer therapies fail cannot be attributed to intrinsic or acquired drug resistance alone. Furthermore, there are inconsistent reports of hypoxia-induced kinase activities in different cancer cell-lines, where increase, decreases, or no change has been observed. Thus, we asked, why are there widely contrasting results in kinase activity under hypoxia in different cancer cell-lines and how does hypoxia play a role in anti-cancer drug sensitivity? RESULTS: We took a modeling approach to address these questions by analyzing the model simulation to explain why hypoxia driven signals can have dissimilar impact on tumor growth and alter the efficacy of anti-cancer drugs. Repeated simulations with varying concentrations of biomolecules followed by decision tree analysis reveal that the highly differential effects among heterogeneous subpopulation of tumor cells could be governed by varying concentrations of just a few key biomolecules. These biomolecules include activated serine/threonine-specific protein kinases (pRAF), mitogen-activated protein kinase kinase (pMEK), protein kinase B (pAkt), or phosphoinositide-4,5-bisphosphate 3-kinase (pPI3K). Additionally, the ratio of activated extracellular signal-regulated kinases (pERK) or pAkt to its respective total was a key factor in determining the sensitivity of pERK or pAkt to hypoxia. CONCLUSION: This work offers a mechanistic insight into how hypoxia can affect the efficacy of anti-cancer drug that targets tumor signaling and provides a framework to identify the types of tumor cells that are either sensitive or resistant to anti-cancer therapy.


Assuntos
Hipóxia/patologia , Neoplasias/patologia , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Teóricos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases raf/metabolismo
13.
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
14.
Pathol Res Pract ; 215(10): 152609, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31488317

RESUMO

BACKGROUND: Breast cancer is the most frequent carcinoma in females, which could be classified to 4 subtypes and the current treatment is still far from satisfactory. In this study, we explored the effects of autophagy inhibition on certain subtypes of breast cancer and the molecular mechanism underlying the different response for breast cancer subtypes initially. METHODS: Autophagy inhibitor hydroxychloroquine (HCQ) was used to identify the sensitivity of breast cancer subtypes to autophagy inhibition in the present study. Cell proliferation and cell invasion were assessed by Cell Counting Kit-8 assay (CCK-8) and transwell assay, respectively. Immunofluorescence staining and western blotting were applied to evaluate cell autophagy. In addition, levels of Ras/Raf/ERK signaling pathway were evaluated by western blotting. RESULTS: Our results showed that HCQ treatment suppressed breast cancer cell proliferation and migration in especially SUM-190 cells, which was most sensitive. Furthermore, HCQ inhibited cell autophagy in breast cancer cells by regulating levels of p62, LC3-I and LC3-II. Moreover, the expression of Ras was significant lower than other breast cancer cells. HCQ treatment markedly inhibited the activation of Ras/Raf/ERK signaling in SUM190 cells. CONCLUSION: To conclude, basal-like breast cancer represented by SUM-190 cells may be most sensitive to HCQ induced autophagy inhibition and the mechanism might be relative to Ras/Raf/ERK signaling pathway.


Assuntos
Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Hidroxicloroquina/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hidroxicloroquina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases raf/metabolismo , Proteínas ras/metabolismo
15.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561471

RESUMO

JC polyomavirus (JCPyV), a ubiquitous human pathogen, is the etiological agent of the fatal neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Like most viruses, JCPyV infection requires the activation of host-cell signaling pathways in order to promote viral replication processes. Previous works have established the necessity of the extracellular signal-regulated kinase (ERK), the terminal core kinase of the mitogen-activated protein kinase (MAPK) cascade (MAPK-ERK) for facilitating transcription of the JCPyV genome. However, the underlying mechanisms by which the MAPK-ERK pathway becomes activated and induces viral transcription are poorly understood. Treatment of cells with siRNAs specific for Raf and MAP kinase kinase (MEK) targets proteins in the MAPK-ERK cascade, significantly reducing JCPyV infection. MEK, the dual-specificity kinase responsible for the phosphorylation of ERK, is phosphorylated at times congruent with early events in the virus infectious cycle. Moreover, a MAPK-specific signaling array revealed that transcription factors downstream of the MAPK cascade, including cMyc and SMAD4, are upregulated within infected cells. Confocal microscopy analysis demonstrated that cMyc and SMAD4 shuttle to the nucleus during infection, and nuclear localization is reduced when ERK is inhibited. These findings suggest that JCPyV induction of the MAPK-ERK pathway is mediated by Raf and MEK and leads to the activation of downstream transcription factors during infection. This study further defines the role of the MAPK cascade during JCPyV infection and the downstream signaling consequences, illuminating kinases as potential therapeutic targets for viral infection.


Assuntos
Interações Hospedeiro-Patógeno , Vírus JC/fisiologia , Sistema de Sinalização das MAP Quinases , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Fatores de Transcrição/metabolismo , Biomarcadores , Células Cultivadas , Resistência à Doença/genética , Suscetibilidade a Doenças , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/genética , Humanos , Infecções por Polyomavirus/genética , Ligação Proteica , Transporte Proteico , Quinases raf/genética , Quinases raf/metabolismo
16.
Folia Biol (Praha) ; 65(2): 70-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31464183

RESUMO

We investigated the detrimental effects of diabetes on myocardium of pregestational streptozotocin (STZ)-diabetic mother rats and their neonates via evaluations of oxidative redox, inflammatory and apoptotic pathways, also aiming to characterize whether calcitriol and/or pomegranate peel extract confer myocardial protection in hyperglycaemic dams and their foetuses via modulation of the Raf/MEK/ERK cascade. Sixty Sprague-Dawley female rats were randomized into five groups (N = 12): control, diabetic, diabetic treated with calcitriol and/or pomegranate peel extract (PPE), and mated with non-diabetic healthy males. After confirmation of pregnancy, treatments were kept until gestational day (E-18). Serum and cardiac tissues of mothers and foetuses were collected and processed for biochemical, histopathological, and molecular assessments. We observed that, compared to the control, diabetic mothers showed dramatically increased hyperglycaemia and hyperlipidaemia associated with decreased myocardial functions and disrupted maternal performance. Also, diabetic mothers and their neonates exhibited elevated levels of myocardial injury (troponin I, endothelin 1, creatine kinase-MB, lactate dehydrogenase), with increased pro-inflammatory cytokines (interleukin 1, interleukin 1ß, transforming growth factor ß) and oxidative redox. Concurrently, the MAPK pathway was significantly down-regulated with increased myocardial apoptotic activity. Furthermore, mRNA expression of angiogenic and fibrotic markers was significantly increased. Paradoxically, calcitriol and/or pomegranate peel extract alleviated these diabetic myocardial insults and normalized the aforementioned assayed parameters. Our findings hypothesized that calcitriol and/or pomegranate peel extract exerted cardioameliorative impacts due to their unique anti-oxidative and anti-inflammatory properties, and thus may be a promising treatment that directly targets the secondary myocardial complications of diabetes in dams and their offspring.


Assuntos
Apoptose , Calcitriol/uso terapêutico , Cardiomiopatias/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Sistema de Sinalização das MAP Quinases , Extratos Vegetais/uso terapêutico , Romã (Fruta)/química , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Cardiomiopatias/complicações , Cardiomiopatias/patologia , Caspase 3/metabolismo , Citocinas/metabolismo , Dano ao DNA , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Feminino , Feto/patologia , Fibrose , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Miocárdio/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Reprodução , Espectroscopia de Infravermelho com Transformada de Fourier , Estreptozocina , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologia , Vitamina D/uso terapêutico , Quinases raf/metabolismo
17.
Artif Cells Nanomed Biotechnol ; 47(1): 3265-3271, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31368817

RESUMO

Luteolin is a representative of natural flavonoid that has anti-tumour properties. This study designed to check its impact on breast cancer and the underlying mechanisms. MDA-MB-453 and MCF-7 cells were administrated with luteolin and the following techniques were carried out: CCK-8 assay, FITC-PI double-staining and Western blot. qRT-PCR analysis was utilized to see the effects of luteolin on miR-203 expression. Besides, miR-203 expression was silenced by transfection with specific inhibitor. Luteolin remarkably declined MDA-MB-453 and MCF-7 cells viability and accelerated apoptosis which accompanied by Bax up-regulation, Bcl-2 down-regulation and Caspase-3 cleavage. Also, luteolin impeded TGFß1-induced EMT, as evidenced by the decreased levels of Vimentin, Zeb1 and N-cadherin, as well as the increased level of E-cadherin. miR-203 was highly expressed in 22 pair of breast cancer tissues than the matched paracancerous tissues. Luteolin could elevate miR-203 level. Besides, luteolin's anti-tumour effects were partially eliminated by miR-203 silence. Further, luteolin inhibited Ras/Raf/MEK/ERK signalling, while the inhibitory effects were flattened by miR-203 silence. Luteolin significantly reduced breast cancer cells growth and EMT. Luteolin exerted its anti-tumour effects possibly involved the elevated expression of miR-203 and the inhibited Ras/Raf/MEK/ERK signalling.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Luteolina/farmacologia , MicroRNAs/genética , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células MCF-7 , Quinases raf/metabolismo , Proteínas ras/metabolismo
18.
Expert Opin Ther Pat ; 29(9): 675-688, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31370713

RESUMO

Introduction: RAF kinase inhibitors block and regulate RAS/RAF/MEK/ERK signaling, which is a key to tumor treatment. At present, although RAF kinase inhibitors have good efficacy, there are few such drugs with low toxicity, and thus, it is urgent to find novel RAF kinase inhibitors associated with higher activity and fewer adverse reactions. This review highlights the anti-tumor effects of several published RAF kinase inhibitors and might be helpful in providing new ideas for the development of novel drug candidates in the future. Areas covered: This article covers the pertinent literature published on RAF kinase inhibitors from 2010 to 2018, as well as the potential use of these compounds as therapeutics for cancer. Expert opinion: To date, many RAF kinase inhibitors with different structures have been studied, many of which have prominent inhibitory activities toward RAF kinase. Further, the specificity of these drugs offers hope for the targeted therapy of tumors. Although RAF kinase inhibition has achieved promising results for the treatment of many cancers, overcoming limitations associated with drug resistance and safety comprises a new direction for the optimization and improvement of RAF kinase inhibitors.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Quinases raf/antagonistas & inibidores , Animais , Desenvolvimento de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Terapia de Alvo Molecular , Neoplasias/enzimologia , Patentes como Assunto , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases raf/metabolismo
19.
J Mater Sci Mater Med ; 30(8): 87, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31325047

RESUMO

Previous studies have demonstrated the significant roles of simvastatin (SVA) and oxysterols in the osteogenesis process. In this study, we evaluate the effect of a combination of SVA and 20(S)-hydroxycholesterol (20(S)OHC) on the cell viability and osteogenic differentiation of bone marrow stromal cells (BMSCs). After treatment with a control vehicle, SVA (0.025, 0.10, 0.25 or 1.0 µM), 20(S)OHC (5 µM), or a combination of both (0.25 µM SVA + 5 µM 20(S)OHC), the proliferation, apoptosis, ALP activity, mineralization, osteogenesis-related gene expression and Raf/MEK/ERK signaling activity in BMSCs were measured. Our results showed that high concentrations of SVA (0.25 and 1.0 µM) enhanced osteogenesis-related genes expression while attenuating cell viability. The addition of 5 µM 20(S)OHC induced significantly higher proliferative activity, which neutralized the inhibitory effect of SVA on the viability of BMSCs. Moreover, compared to supplementation with only one of the additives, combined supplementation with both SVA and 20(S)OHC induced significantly enhanced ALP activity, calcium sedimentation, osteogenesis-related genes (ALP, OCN and BMP-2) expression and Raf/MEK/ERK signaling activity in BMSCs; these enhancements were attenuated by treatment with the inhibitor U0126, indicating a significant role of Raf/MEK/ERK signaling in mediating the synergistically enhanced osteogenic differentiation of BMSCs by combined SVA and 20(S)OHC treatment. Additionally, histological examination confirmed a synergistic effect of SVA and 20(S)OHC on enhancing bone regeneration in a rabbit calvarial defect model. This newly developed SVA/20(S)OHC formulation may be used as an osteoinductive drug to enhance bone healing.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Sinvastatina/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Regeneração Óssea/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Células-Tronco Mesenquimais/fisiologia , Osteogênese/genética , Coelhos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Quinases raf/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(27): 13330-13339, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31213532

RESUMO

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases raf/química , Quinases raf/metabolismo , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Edição de Genes , Técnicas de Inativação de Genes , Humanos , Fosforilação , Multimerização Proteica , Proteínas ras/metabolismo
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