Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Cell Death Dis ; 14(7): 436, 2023 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-37454104

RESUMO

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.


Assuntos
Autofagia , Proteínas rab de Ligação ao GTP , Fosforilação/fisiologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação , Autofagia/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(28): e2113465119, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35867735

RESUMO

The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant KrasG12D and mutant Trp53172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of KrasG12D/+ and Trp53172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic KrasG12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7+/- tumors have comparatively lower levels of succinate, and that cells derived from Atg7+/- tumors are also less invasive than those from Atg7+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7+/- cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy.


Assuntos
Proteína 7 Relacionada à Autofagia , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Camundongos , Mutação , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Succinatos/metabolismo , Succinatos/farmacologia
3.
Proc Natl Acad Sci U S A ; 119(26): e2111506119, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35737835

RESUMO

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.


Assuntos
Fucosidose , Macroautofagia , Polissacarídeos , Animais , Fucosidose/genética , Fucosidose/metabolismo , Lisossomos/metabolismo , Macroautofagia/fisiologia , Camundongos , Polissacarídeos/metabolismo , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/metabolismo
4.
FEBS J ; 289(13): 3752-3769, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35060334

RESUMO

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic material to lysosomes for degradation. The process preserves cellular integrity by removing damaged cellular constituents and can promote cell survival by providing substrates for energy production during hiatuses of nutrient availability. The process is also highly responsive to other forms of cellular stress. For example, DNA damage can induce autophagy and this involves up-regulation of the Damage-Regulated Autophagy Modulator-1 (DRAM-1) by the tumor suppressor p53. DRAM-1 belongs to an evolutionarily conserved protein family, which has five members in humans and we describe here the initial characterization of two members of this family, which we term DRAM-4 and DRAM-5 for DRAM-Related/Associated Member 4/5. We show that the genes encoding these proteins are not regulated by p53, but instead are induced by nutrient deprivation. Similar to other DRAM family proteins, however, DRAM-4 principally localizes to endosomes and DRAM-5 to the plasma membrane and both modulate autophagy flux when over-expressed. Deletion of DRAM-4 using CRISPR/Cas-9 also increased autophagy flux, but we found that DRAM-4 and DRAM-5 undergo compensatory regulation, such that deletion of DRAM-4 does not affect autophagy flux in the absence of DRAM-5. Similarly, deletion of DRAM-4 also promotes cell survival following growth of cells in the absence of amino acids, serum, or glucose, but this effect is also impacted by the absence of DRAM-5. In summary, DRAM-4 and DRAM-5 are nutrient-responsive members of the DRAM family that exhibit interconnected roles in the regulation of autophagy and cell survival under nutrient-deprived conditions.


Assuntos
Proteínas de Membrana , Proteína Supressora de Tumor p53 , Apoptose/fisiologia , Autofagia/fisiologia , Sobrevivência Celular/genética , Humanos , Proteínas de Membrana/metabolismo , Nutrientes , Proteína Supressora de Tumor p53/genética
5.
Sci Adv ; 7(23)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34088666

RESUMO

Hepatocellular carcinoma (HCC) is driven by repeated rounds of inflammation, leading to fibrosis, cirrhosis, and, ultimately, cancer. A critical step in HCC formation is the transition from fibrosis to cirrhosis, which is associated with a change in the liver parenchyma called ductular reaction. Here, we report a genetically engineered mouse model of HCC driven by loss of macroautophagy and hemizygosity of phosphatase and tensin homolog, which develops HCC involving ductular reaction. We show through lineage tracing that, following loss of autophagy, mature hepatocytes dedifferentiate into biliary-like liver progenitor cells (ductular reaction), giving rise to HCC. Furthermore, this change is associated with deregulation of yes-associated protein and transcriptional coactivator with PDZ-binding motif transcription factors, and the combined, but not individual, deletion of these factors completely reverses the dedifferentiation capacity and tumorigenesis. These findings therefore increase our understanding of the cell of origin of HCC development and highlight new potential points for therapeutic intervention.

6.
Mol Cell ; 76(1): 163-176.e8, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31492633

RESUMO

Sensing nutrient availability is essential for appropriate cellular growth, and mTORC1 is a major regulator of this process. Mechanisms causing mTORC1 activation are, however, complex and diverse. We report here an additional important step in the activation of mTORC1, which regulates the efflux of amino acids from lysosomes into the cytoplasm. This process requires DRAM-1, which binds the membrane carrier protein SCAMP3 and the amino acid transporters SLC1A5 and LAT1, directing them to lysosomes and permitting efficient mTORC1 activation. Consequently, we show that loss of DRAM-1 also impacts pathways regulated by mTORC1, including insulin signaling, glycemic balance, and adipocyte differentiation. Interestingly, although DRAM-1 can promote autophagy, this effect on mTORC1 is autophagy independent, and autophagy only becomes important for mTORC1 activation when DRAM-1 is deleted. These findings provide important insights into mTORC1 activation and highlight the importance of DRAM-1 in growth control, metabolic homeostasis, and differentiation.


Assuntos
Aminoácidos/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Metabolismo Energético , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Células 3T3-L1 , Adipócitos/enzimologia , Adipogenia , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema y+L de Transporte de Aminoácidos/genética , Sistema y+L de Transporte de Aminoácidos/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/genética , Glicemia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Insulina/sangue , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Transporte Proteico
7.
Methods Mol Biol ; 1880: 359-374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610710

RESUMO

Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.


Assuntos
Autofagia/genética , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular , Técnicas de Silenciamento de Genes/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção/instrumentação , Transfecção/métodos
8.
Transcription ; 9(2): 131-136, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28980873

RESUMO

Autophagy is an essential cellular process that degrades cytoplasmic organelles and components. Precise control of autophagic activity is achieved by context-dependent signaling pathways. Recent studies have highlighted the involvement of transcriptional programs during autophagic responses to various signals. Here, we summarize the current understanding of the transcriptional regulation of autophagy.


Assuntos
Autofagia , Transcrição Gênica , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ciclo Celular , Código das Histonas , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional
9.
Mol Cell ; 66(4): 517-532.e9, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525743

RESUMO

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.


Assuntos
Autofagia , Carcinoma Ductal Pancreático/metabolismo , Lisossomos/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Regulação para Baixo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Agregados Proteicos , Ligação Proteica , Proteólise , Interferência de RNA , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção
10.
Front Oncol ; 7: 28, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28316954

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the few cancer types where the 5-year survival rate shows no improvement. Despite conflicting evidence, the majority of data points to an essential role for autophagy in PDAC growth and survival, in particular constitutively activated autophagy, can provide crucial fuel to PDAC tumor cells in their nutrient-deprived environment. Autophagy, which is required for cell homeostasis, can both suppress and promote tumorigenesis and tumor survival in a context-dependent manner. Protein by protein, the mystery of how PDAC abuses the cell's homeostasis system for its malignant growth has recently begun to be unraveled. In this review, we focus on how autophagy is responsible for growth and development of PDAC tumors and where autophagy and the mechanisms controlling it fit into PDAC metabolism. Understanding the range of pathways controlling autophagy and their interplay in PDAC could open the way for new therapeutic avenues.

11.
Proc Natl Acad Sci U S A ; 112(3): 773-8, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25568088

RESUMO

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.


Assuntos
Autofagia , Reparo do DNA/genética , Genes Letais , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Recombinação Homóloga , Camundongos , Reação em Cadeia da Polimerase em Tempo Real
12.
Mol Cell ; 50(3): 394-406, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23603120

RESUMO

Tumor cells undergo changes in metabolism to meet their energetic and anabolic needs. It is conceivable that mechanisms exist to sense these changes and link them to pathways that eradicate cells primed for cancer development. We report that the tumor suppressor p53 activates a cell death priming mechanism that senses extracellular adenosine. Adenosine, the backbone of ATP, accumulates under conditions of cellular stress or altered metabolism. We show that its receptor, A2B, is upregulated by p53. A2B expression has little effect on cell viability, but ligand engagement activates a caspase- and Puma-dependent apoptotic response involving downregulation of antiapoptotic Bcl-2 proteins. Stimulation of A2B also significantly enhances cell death mediated by p53 and upon accumulation of endogenous adenosine following chemotherapeutic drug treatment and exposure to hypoxia. Since extracellular adenosine also accumulates within many solid tumors, this distinct p53 function links programmed cell death to both a cancer- and therapy-associated metabolic change.


Assuntos
Adenosina/genética , Adenosina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/genética , Caspases/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação para Baixo/genética , Células HCT116 , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor A2B de Adenosina/genética , Receptor A2B de Adenosina/metabolismo , Regulação para Cima/genética
13.
Biochem Soc Trans ; 40(1): 94-100, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22260672

RESUMO

There is an increasing body of evidence demonstrating a critical role for the bioactive lipid S1P (sphingosine 1-phosphate) in cancer. S1P is synthesized and metabolized by a number of enzymes, including sphingosine kinase, S1P lyase and S1P phosphatases. S1P binds to cell-surface G-protein-coupled receptors (S1P1-S1P5) to elicit cell responses and can also regulate, by direct binding, a number of intracellular targets such as HDAC (histone deacetylase) 1/2 to induce epigenetic regulation. S1P is involved in cancer progression including cell transformation/oncogenesis, cell survival/apoptosis, cell migration/metastasis and tumour microenvironment neovascularization. In the present paper, we describe our research findings regarding the correlation of sphingosine kinase 1 and S1P receptor expression in tumours with clinical outcome and we define some of the molecular mechanisms underlying the involvement of sphingosine kinase 1 and S1P receptors in the formation of a cancer cell migratory phenotype. The role of sphingosine kinase 1 in the acquisition of chemotherapeutic resistance and the interaction of S1P receptors with oncogenes such as HER2 is also reviewed. We also discuss novel aspects of the use of small-molecule inhibitors of sphingosine kinase 1 in terms of allosterism, ubiquitin-proteasomal degradation of sphingosine kinase 1 and anticancer activity. Finally, we describe how S1P receptor-modulating agents abrogate S1P receptor-receptor tyrosine kinase interactions, with potential to inhibit growth-factor-dependent cancer progression.


Assuntos
Lisofosfolipídeos/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Oncogenes , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/metabolismo
15.
Cell Cycle ; 9(20): 4050-1, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20980805
16.
J Biol Chem ; 285(50): 38841-52, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-20926375

RESUMO

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Tiazóis/farmacologia , Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Densitometria , Feminino , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Ubiquitina/química
17.
Am J Pathol ; 177(5): 2205-15, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20889557

RESUMO

Various studies in cell lines have previously demonstrated that sphingosine kinase 1 (SK1) and extracellular signal-regulated kinase 1/2 (ERK-1/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of sphingosine 1-phosphate (S1P) receptors 1, 2, and 3 (ie, S1P1, S1P2, and S1P3), SK1, and ERK-1/2 expression levels. Expression levels of both SK1 and ERK-1/2 were already available for the cohort, and S1P1, S1P2, and S1P3 levels were established by immunohistochemical analysis. High membrane S1P1 expression was associated with shorter time to recurrence (P=0.008). High cytoplasmic S1P1 and S1P3 expression levels were also associated with shorter disease-specific survival times (P=0.036 and P=0.019, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK1 and ERK-1/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK1 and cytoplasmic ERK-1/2 (P=0.00008), with a difference in recurrence time of 10.5 years. Similarly, high cytoplasmic S1P1 and cytoplasmic ERK-1/2 expression levels (P=0.004) and high cytoplasmic S1P3 expression and cytoplasmic ERK-1/2 expression levels (P=0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK1, S1P1, and/or S1P3 and ERK-1/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.


Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Tamoxifeno/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Lisofosfolipídeos/metabolismo , Pessoa de Meia-Idade , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Lisoesfingolipídeo/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Taxa de Sobrevida
18.
J Biol Chem ; 285(46): 35957-66, 2010 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-20837468

RESUMO

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P(4)) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P(2/4) antagonist and reduced by siRNA knockdown of S1P(4). Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P(4) agonist, stimulated ERK-1/2 activation in an S1P(4)- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P(1,3,4,5) but not S1P(2) stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P(4) may have an important role in breast cancer progression.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Cloridrato de Fingolimode , Células HEK293 , Humanos , Lisofosfolipídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação/efeitos dos fármacos , Propilenoglicóis/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Interferência de RNA , Receptor ErbB-2/genética , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologia
19.
Mol Cell Biol ; 30(15): 3827-41, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20516217

RESUMO

We demonstrate here a new concept termed "oncogene tolerance" whereby human EGF receptor 2 (HER2) increases sphingosine kinase 1 (SK1) expression in estrogen receptor-positive (ER(+)) MCF-7 HER2 cells and SK1, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK1 expression also limits p21-activated protein kinase 1 (p65 PAK1) and extracellular signal regulated kinase 1/2 (ERK-1/2) signaling. Sphingosine 1-phosphate signaling via S1P(3) is also altered in MCF-7 HER2 cells. In this regard, S1P binding to S1P(3) induces a migratory phenotype via an SK1-dependent mechanism in ER(+) MCF-7 Neo cells, which lack HER2. This involves the S1P stimulated accumulation of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S1P failed to promote redistribution of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S1P when SK1 expression had been knocked down with a specific siRNA or when recombinant PAK1 was ectopically overexpressed. Thus, the HER2-dependent increase in SK1 expression functions to desensitize the S1P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER1-3/SK1 expression ratio in their ER(+) breast cancer tumors compared to patients that have a high HER1-3/SK1 expression ratio.


Assuntos
Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Receptores de Estrogênio/metabolismo , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptor alfa de Estrogênio , Humanos , Lisofosfolipídeos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/farmacologia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool) , Ligação Proteica/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Estrogênio/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingosina/análogos & derivados , Quinases Ativadas por p21
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...