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Introduction: Breast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties. Methods: Triple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice. Results: Here, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells. Discussion: These results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically.
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Purpose: Proton pump inhibitors (PPIs) are the first-line therapy for gastroesophageal reflux disorder (GERD). Unlike conventional PPIs, non-enteric coated PPIs with antacid salt enable a faster acid suppression through the rapid absorption of the PPI. YPI-011 is a newly developed fixed-dose combination of a rabeprazole with sodium bicarbonate (NaHCO3). This study compared the pharmacokinetics (PKs) and pharmacodynamics (PDs) of YPI-011 to the conventional enteric-coated rabeprazole (Pariet®). Materials and Methods: A randomized, open-label, two-treatment, two-sequence crossover study was conducted with two different doses (10 and 20 mg) and 44 subjects in each group. They randomly received either a test or reference treatment for 7 days in the first period and the other treatment in the second period. Blood samples for the PK analysis were taken after the single- and multiple-dose. Intragastric pH monitoring for the PD analysis was implemented for baseline and after the single- and multiple-dose. Results: Gastric acid suppression evaluated by the percentage decrease from baseline in the integrated gastric acidity for a 24-hour interval after the multiple-dose was similar between the treatments in both dose groups. The systemic exposure of rabeprazole at steady state after the multiple-dose was also similar between the treatments in both dose groups. The time to reach the maximum rabeprazole concentration was faster in the test treatment. The PK-PD relationship of PPI is well known, and the faster absorption of rabeprazole resulted in a more rapid mode of action in acid suppression. Conclusion: The fixed dose combination of rabeprazole with NaHCO3 showed a faster absorption and consequently, a more rapid gastric acid suppression with a similar systemic exposure of rabeprazole at steady state compared to the conventional enteric-coated rabeprazole.
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Antiulcerosos , Inibidores da Bomba de Prótons , Humanos , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol , Bicarbonato de Sódio , Estudos Cross-Over , Antiulcerosos/farmacologia , Concentração de Íons de HidrogênioRESUMO
Retinoic acid receptor responder 1 (RARRES1) is among the most commonly methylated loci in multiple cancers. RARRES1 regulates mitochondrial and fatty acid metabolism, stem cell differentiation, and survival of immortalized cell lines in vitro. Here, we created constitutive Rarres1 knockout (Rarres1-/-) mouse models to study RARRES1 function in vivo. Rarres1-/- embryonic fibroblasts regulated tubulin glutamylation, cell metabolism, and survival, recapitulating RARRES1 function in immortalized cell lines. In two mouse strains, loss of Rarres1 led to a markedly increased dose-dependent incidence of follicular lymphoma (FL). Prior to lymphoma formation, Rarres1-/- B cells have compromised activation, maturation, differentiation into antibody-secreting plasma cells, and cell cycle progression. Rarres1 ablation increased B cell survival and led to activation of the unfolded protein response (UPR) and heat shock response (HSR). Rarres1 deficiency had differential effects on cellular metabolism, with increased bioenergetic capacity in fibroblasts, and minor effects on bioenergetics and metabolism in B cells. These findings reveal that RARRES1 is a bona fide tumor suppressor in vivo and the deletion in mice promotes cell survival, and reduces B cell differentiation with B cell autonomous and non-autonomous functions.
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Genes Supressores de Tumor , Proteínas de Membrana , Animais , Diferenciação Celular/genética , Linhagem Celular , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.
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Senescência Celular/efeitos dos fármacos , Quimiocinas CC/metabolismo , Peróxido de Hidrogênio/farmacologia , NF-kappa B/metabolismo , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocina CCL7/antagonistas & inibidores , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/genética , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteína-Arginina Desiminase do Tipo 2/antagonistas & inibidores , Proteína-Arginina Desiminase do Tipo 2/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
[This corrects the article DOI: 10.18632/oncotarget.26600.].
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Fibroblast growth factor receptor 2 (FGFR2) is a membrane-spanning tyrosine kinase that mediates FGF signaling. Various FGFR2 alterations are detected in breast cancer, yet it remains unclear if activation of FGFR2 signaling initiates tumor formation. In an attempt to answer this question, a mouse model berrying an activation mutation of FGFR2 (FGFR2-S252W) in the mammary gland is generated. It is found that FGF/FGFR2 signaling drives the development of triple-negative breast cancer accompanied by epithelial-mesenchymal transition that is regulated by FGFR2-STAT3 signaling. It is demonstrated that FGFR2 suppresses BRCA1 via the ERK-YY1 axis and promotes tumor progression. BRCA1 knockout in the mammary gland of the FGFR2-S252W mice significantly accelerated tumorigenesis. It is also shown that FGFR2 positively regulates PD-L1 and that a combination of FGFR2 inhibition and immune checkpoint blockade kills cancer cells. These data suggest that the mouse models mimic human breast cancers and can be used to identify actionable therapeutic targets.
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Proteína BRCA1/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Neoplasias de Mama Triplo Negativas/terapia , Animais , Antígeno B7-H1/metabolismo , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Imunoterapia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Fator de Transcrição YY1/metabolismoRESUMO
Atopic dermatitis (AD) is an inflammatory skin disease in which type 2 allergic inflammation plays a critical role. In this study, the anti-inflammatory effect of conditioned media from human umbilical cord blood-derived mesenchymal stem cells (USC-CM) was investigated in order to apply it as an effective treatment with a low risk of side effects that can overcome the limitations of AD treatment which is currently in use. We found that USC-CM has various growth factors and cytokines associated with anti-inflammatory effect. RT-PCR and ELISA analysis showed that USC-CM inhibited the levels of type 2 cytokine and chemokine Thymus and activation-regulated chemokine (TARC), TNF-α and IL-6 in TNF-α/IFN-γ-stimulated HaCaT cells. In addition, USC-CM inhibited IL-4 and IL-13 levels in Th2 cells. Therefore, the results of our study demonstrated that USC-CM has anti-inflammatory effect in TNF-α/IFN-γ-stimulated HaCaT cells which associated with the inhibition of the immunoglobulin (IgE) secretion by activating B cell line. Our In vivo results showed that when the USC-CM was applied to lesions of patients with the mild AD for 4 weeks, the skin barrier was strengthened by increasing the level of Corneometer and decreasing the value of transepidermal water loss (TEWL). In conclusion, the results suggest that USC-CM may have therapeutic effect for AD as cosmetics and drug materials.
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Meios de Cultivo Condicionados/farmacologia , Dermatite Atópica/terapia , Células-Tronco Mesenquimais/citologia , Pele/patologia , Linhagem Celular , Quimiocinas/imunologia , Citocinas/imunologia , Dermatite Atópica/imunologia , Feminino , Sangue Fetal/citologia , Humanos , Imunidade/imunologia , Masculino , Pele/imunologia , Resultado do Tratamento , Perda Insensível de Água/fisiologiaAssuntos
Antígenos CD/biossíntese , Ciclo-Oxigenase 2/biossíntese , Melanoma/etiologia , Neoplasias Cutâneas/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Correlação de Dados , Feminino , Humanos , Masculino , Melanoma/mortalidade , Pessoa de Meia-Idade , Prognóstico , Neoplasias Cutâneas/mortalidade , Taxa de Sobrevida , Adulto JovemAssuntos
Toxinas Botulínicas Tipo A/farmacologia , Técnicas Cosméticas , Músculos Faciais/efeitos dos fármacos , Fármacos Neuromusculares/farmacologia , Glândula Parótida/efeitos dos fármacos , Adulto , Músculos Faciais/diagnóstico por imagem , Feminino , Humanos , Injeções , Glândula Parótida/diagnóstico por imagem , República da Coreia , Tomografia Computadorizada por Raios XRESUMO
An Al3+-based metal-organic framework (MOF), CAU-11-COOH, with a V-shaped ligand, DPSDA (3,3'-4,4'-diphenylsulfonetetracarboxylic dianhydride), was prepared using the solvothermal method, and was characterized using powder X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, elemental analysis, thermogravimetric analysis, Brunauer-Emmett-Teller analysis, and CO2 adsorption. The catalytic efficiency of CAU-11-COOH was investigated in the solvent-free cycloaddition of carbon dioxide with epoxides, which yielded five-membered cyclic carbonates under mild reaction conditions. CAU-11-COOH with a co-catalyst, tetrabutylammonium bromide (TBAB), gave higher than 98% yield of epichlorohydrin carbonate at 80 °C without a solvent. A plausible reaction mechanism in which the Lewis acidic metal center, an uncoordinated carboxyl group, and a nucleophilic bromide anion operate synergistically is proposed. The CAU-11-COOH catalysts were found to exhibit high thermal stability and could be reused more than four times without any significant reduction in activity.
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Desensitization, signaling, and trafficking of G-protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, ß-arrestins (ßarrs). The two isoforms of ßarrs (ßarr1 and 2) share a high degree of sequence and structural similarity; still, however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of ßarr isoforms is still lacking. By using a set of complementary approaches, including antibody-fragment-based conformational sensors, we discover structural differences between ßarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain-swapped chimeras of ßarrs display robust complementation in functional assays, thereby linking the structural differences between receptor-bound ßarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of ßarr isoforms to drive distinct functional outcomes and underscore the importance of integrating this aspect in the current framework of biased agonism.
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beta-Arrestinas/química , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , beta-Arrestinas/genética , beta-Arrestinas/metabolismoRESUMO
Information contained in the structure of extracellular ligands is transmitted across the cell membrane through allosterically induced changes in G protein-coupled receptor (GPCR) conformation that occur upon ligand binding. These changes, in turn, are imprinted upon intracellular effectors like arrestins and help determine which of its many functions are performed. Intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET), in which both the fluorescence donor and acceptor are contained within the same protein, can be used to report on activation-induced changes in protein conformation. Here, we describe a method using a series of Rluc-arrestin3-FlAsH-BRET biosensors to measure stimulus-induced changes in arrestin conformation in live cells. Each Rluc-arrestin3-FlAsH-BRET construct contains an N-terminal Renilla luciferase fluorescence donor that excites a fluorescent arsenical targeted to a different position within the protein by mutational insertion of a tetracysteine tag motif. Changes in net BRET upon GPCR stimulation can thus be viewed from multiple vantage points within the protein and used to develop an arrestin3 "conformational signature" that is receptor- and ligand-specific. This method can be used to determine how differences in GPCR and ligand structure influence information transfer across the plasma membrane and to classify GPCRs and/or ligands based on their capacity to induce different arrestin3 activation modes.
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Arrestina/metabolismo , Arsênio/química , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Fluoresceína/química , Análise de Dados , Células HEK293 , Humanos , Reprodutibilidade dos TestesRESUMO
RARRES1, a retinoic acid regulated carboxypeptidase inhibitor associated with fatty acid metabolism, stem cell differentiation and tumorigenesis is among the most commonly methylated loci in multiple cancers but has no known mechanism of action. Here we show that RARRES1 interaction with cytoplasmic carboxypeptidase 2 (CCP2) inhibits tubulin deglutamylation, which in turn regulates the mitochondrial voltage dependent anion channel (VDAC1), mitochondrial membrane potential, AMPK activation, energy balance and metabolically reprograms cells and zebrafish to a more energetic and anabolic phenotype. Depletion of RARRES1 also increases expression of stem cell markers, promotes anoikis, anchorage independent growth and insensitivity to multiple apoptotic stimuli. As depletion of CCP2 or inhibition of VDAC1 reverses the effects of RARRES1 depletion on energy balance and cell survival we conclude that RARRES1 modulation of CCP2-modulated tubulin-mitochondrial VDAC1 interactions is a fundamental regulator of cancer and stem cell metabolism and survival.
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Linfoma Cutâneo de Células T/patologia , Linfoma Cutâneo de Células T/fisiopatologia , Linfoma de Células T Periférico/patologia , Linfoma de Células T Periférico/fisiopatologia , Neoplasias Cutâneas/patologia , Adulto , Distribuição por Idade , Idoso , Biópsia por Agulha , Bases de Dados Factuais , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Incidência , Linfoma Cutâneo de Células T/epidemiologia , Linfoma de Células T Periférico/epidemiologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Prognóstico , República da Coreia , Estudos Retrospectivos , Medição de Risco , Distribuição por Sexo , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/fisiopatologiaRESUMO
G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. ß-Arrestins (ßArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete ßArr1/2 and G proteins have cast doubt on the role of ß-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of ßArr1/2 and reconstitution with ßArr1/2 in three different parental and CRISPR-derived ßArr1/2 knockout HEK293 cell pairs to assess the effect of ßArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for ßArr2 or ßArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For ß2 adrenergic receptors (ß2ARs) and ß1ARs, ßArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with ßArr1/2. Loss of desensitization and receptor internalization in CRISPR ßArr1/2 knockout cells caused ß2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing ßArr1/2. These data suggest that ßArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from ßArr1/2- or G protein-deleted cells to GPCR behavior in native systems.
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Sistemas CRISPR-Cas , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Ativação Enzimática , Deleção de Genes , Edição de Genes , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , Receptores Adrenérgicos beta 2/metabolismoRESUMO
HDL normally transports about 50-70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.