RESUMEN
Chronic rhinosinusitis with nasal polyps is a widespread pathology characterized by persistent inflammation of nasal and paranasal mucosa. Although it represents one of the most frequent diseases of the nasal cavities, its etiology is still not completely elucidated. There is evidence suggesting that the Notch signaling, a highly conserved intercellular pathway known to regulate many cellular processes, including inflammation, is implicated in nasal polyps formation. The purpose of this study was to investigate the expression of genes of the Notch pathway in nasal polyps from patients with chronic rhinosinusitis. Nasal polyps and adjacent mucosa tissue were obtained from 10 patients. RNA was analyzed by quantitative reverse transcriptase-polymerase chain reaction for the expression level of (1) Notch pathway components such as receptors (NOTCH1-4), ligands (DLL4, JAGGED-1), and target genes (HEY1, 2, and HES1) and (2) genes providing information on the pathogenesis of polyposis (C-MYC and SCGB1A1) and on eosinophils content (CCL26, IL5, and SAA2). We report a Notch-driven gene expression pattern in nasal polyps which correlates with the expression of genes highly expressed in eosinophils, whose presence is an important parameter to define the pathophysiologic diversity characterizing nasal polyps. Taken together, our results suggest a role for Notch signaling in the pathophysiology of polyposis. Further studies are needed to elucidate the role of Notch in nasal polyps formation and to establish whether it could represent a novel therapeutic target for this pathology.
Asunto(s)
Pólipos Nasales , Sinusitis , Enfermedad Crónica , Eosinófilos , Humanos , Inflamación , Proyectos PilotoRESUMEN
Ticagrelor is a powerful P2Y12 inhibitor with pleiotropic effects in the cardiovascular system. Consistently, we have reported that in patients with stable coronary artery disease (CAD) and concomitant chronic obstructive pulmonary disease (COPD) who underwent percutaneous coronary intervention (PCI), 1-month treatment with ticagrelor was superior in improving biological markers of endothelial function, compared with clopidogrel. The objective of this study was to investigate the mechanisms underlying these beneficial effects of ticagrelor by conducting molecular analyses of RNA isolated from peripheral blood cells of these patients. We determined mRNAs levels of markers of inflammation and oxidative stress, such as RORγt (T helper 17 cells marker), FoxP3 (regulatory T cells marker), NLRP3, ICAM1, SIRT1, Notch ligands JAG1 and DLL4, and HES1, a Notch target gene. We found that 1-month treatment with ticagrelor, but not clopidogrel, led to increased levels of SIRT1 and HES1 mRNAs. In patients treated with ticagrelor or clopidogrel, we observed a negative correlation among changes in both SIRT1 and HES1 mRNA and serum levels of Epidermal Growth Factor (EGF), a marker of endothelial dysfunction found to be reduced by ticagrelor treatment in our previous study. In conclusion, we report that in stable CAD/COPD patients ticagrelor positively regulates HES1 and SIRT1, two genes playing a protective role in the context of inflammation and oxidative stress. Our observations confirm and expand previous studies showing that the beneficial effects of ticagrelor in stable CAD/COPD patients may be, at least in part, mediated by its capacity to reduce systemic inflammation and oxidative stress.
Asunto(s)
Células Sanguíneas/efectos de los fármacos , Enfermedad de la Arteria Coronaria/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Sirtuina 1/genética , Ticagrelor/farmacología , Factor de Transcripción HES-1/genética , Células Sanguíneas/metabolismo , Células Cultivadas , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal , Sirtuina 1/metabolismo , Factor de Transcripción HES-1/metabolismoRESUMEN
Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/- mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.
Asunto(s)
Aorta/metabolismo , Aterosclerosis/genética , Endotelio Vascular/metabolismo , Proteína KRIT1/genética , Mutación con Pérdida de Función , Animales , Aorta/patología , Apoptosis , Aterosclerosis/metabolismo , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Proteína KRIT1/deficiencia , Proteína KRIT1/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Receptor Notch1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2019.00162.].
RESUMEN
Estrogen regulates a plethora of biological processes, under physiological and pathological conditions, by affecting key pathways involved in the regulation of cell proliferation, fate, survival and metabolism. The Notch receptors are mediators of communication between adjacent cells and are key determinants of cell fate during development and in postnatal life. Crosstalk between estrogen and the Notch pathway intervenes in many processes underlying the development and maintenance of the cardiovascular system. The identification of molecular mechanisms underlying the interaction between these types of endocrine and juxtacrine signaling are leading to a deeper understanding of physiological conditions regulated by these steroid hormones and, potentially, to novel therapeutic approaches to prevent pathologies linked to reduced levels of estrogen, such as coronary heart disease, and cardiotoxicity caused by hormone therapy for estrogen-receptor-positive breast cancer.
Asunto(s)
Enfermedad Coronaria/metabolismo , Estrógenos/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Enfermedad Coronaria/etiología , Enfermedad Coronaria/patología , Humanos , Factores ProtectoresRESUMEN
Notch receptors signaling is required for optimal T-cell activation and function. T-cell receptor (TCR) engagement can activate Notch receptors in T-cells in a ligand-independent fashion. In this study, we examined the role of adenosine A2A receptor (A2AR) signaling pathway in regulating the activity of Notch1 induced by TCR stimulation in CD8+T-cells. A selective A2AR agonist decreased Notch1 protein expression and Notch1 cleavage, and reduced transcripts of Notch1-target genes Hes1 and Myc in activated CD8+T-cells. Inhibition of TCR-induced Notch1 expression by an A2AR agonist was accompanied by increased cAMP concentration and mimicked by forskolin. This effect was associated with reduced IFN-γ and granzyme B production. The effect of an A2AR agonist was abrogated by a selective A2AR antagonist and absent in CD8+T-cells harvested from A2AR-/- mice. Stimulation of A2AR reduced Notch1 receptor levels by inhibiting upstream TCR signals, including ZAP70 phosphorylation, in turn impairing the generation of the active Notch1 intracellular domain (N1ICD). Direct activation of PKC with PMA and ionomycin bypassed A2AR-induced Notch1 inhibition. Overexpression of N1ICD in CD8+T-cells prevented the suppressive effects of an A2AR agonist on proliferation and cytokine release during activation. Our results identify the A2AR signaling pathway as an important regulator of TCR-induced Notch1 receptor activation in CD8+T-cells, and Notch as an important target of the immune suppressive effects of A2AR. We propose a mechanism whereby A2AR impairs CD8 T-cells function through inhibition of Notch1 receptor activation.
RESUMEN
Triple negative breast cancer (TNBC) patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Notch signaling is a key pathway regulating TNBC CSC survival. Treatment of TNBC with PI3K or mTORC1/2 inhibitors results in drug-resistant, Notch-dependent CSC. However, downstream mechanisms and potentially druggable Notch effectors in TNBC CSCs are largely unknown. We studied the role of the AKT pathway and mitochondrial metabolism downstream of Notch signaling in TNBC CSC from cell lines representative of different TNBC molecular subtypes as well as a novel patient-derived model. We demonstrate that exposure of TNBC cells to recombinant Notch ligand Jagged1 leads to rapid AKT phosphorylation in a Notch1-dependent but RBP-Jκ independent fashion. This requires mTOR and IKKα. Jagged1 also stimulates mitochondrial respiration and fermentation in an AKT- and IKK-dependent fashion. Notch1 co-localizes with mitochondria in TNBC cells. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor PF-03084014 in combination with AKT inhibitor MK-2206 or IKK-targeted NF-κB inhibitor Bay11-7082 blocks secondary mammosphere formation from sorted CD90hi or CD44+CD24low (CSCs) cells. A TNBC patient-derived model gave comparable results. Besides mitochondrial oxidative metabolism, Jagged1 also triggers nuclear, NF-κB-dependent transcription of anti-apoptotic gene cIAP-2. This requires recruitment of Notch1, IKKα and NF-κB to the cIAP-2 promoter. Our observations support a model where Jagged1 triggers IKKα-dependent, mitochondrial and nuclear Notch1 signals that stimulate AKT phosphorylation, oxidative metabolism and transcription of survival genes in PTEN wild-type TNBC cells. These data suggest that combination treatments targeting the intersection of the Notch, AKT and NF-κB pathways have potential therapeutic applications against CSCs in TNBC cases with Notch1 and wild-type PTEN expression.
RESUMEN
The transmembrane receptor, Notch1 plays an important role during the differentiation of CD4 T cells into T helper (Th) subsets in the presence of appropriate cytokines, including differentiation into Th1 cells. MicroRNAs have also been shown to be important regulators of immune responses, including negatively regulating cytokine production by Th1 cells. The miR-29 family of microRNAs can act to inhibit tbx21 and ifng transcription, two important pro-inflammatory genes that are abundantly expressed in Th1 cells. Here we show that Notch1 may prime CD4 T cells to be responsive to Th1-polarizing cues through its early repressive effects on the miR-29 family of microRNAs. Using a combination of cell lines and primary cells, we demonstrate that Notch1 can repress miR-29a, miR-29b, and miR-29c transcription through a mechanism that is independent of NF-κB. We further show that this repression is mediated by canonical Notch signaling and requires active Mastermind like (MAML) 1, but this process is superseded by positive regulation of miR-29 in response to IFNγ at later stages of CD4 T cell activation and differentiation. Collectively, our data suggest an additional mechanism by which Notch1 signaling may fine-tune Th1 cell differentiation.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/fisiología , MicroARNs/metabolismo , Receptor Notch1/metabolismo , Células TH1/metabolismo , Animales , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Células 3T3 NIH , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Next generation sequencing (NGS) is becoming increasingly integrated into oncological practice and clinical research. NGS methods have also provided evidence for clonal evolution of cancers during disease progression and treatment. The number of variants associated with response to specific therapeutic agents keeps increasing. However, the identification of novel driver mutations as opposed to passenger (phenotypically silent or clinically irrelevant) mutations remains a major challenge. We conducted targeted exome sequencing of advanced solid tumors from 44 pre-treated patients with solid tumors including breast, colorectal and lung carcinomas, neuroendocrine tumors, sarcomas and others. We catalogued established driver mutations and putative new drivers as predicted by two distinct algorithms. The established drivers we detected were consistent with published observations. However, we also detected a significant number of mutations with driver potential never described before in each tumor type we studied. These putative drivers belong to key cell fate regulatory networks, including potentially druggable pathways. Should our observations be confirmed, they would support the hypothesis that new driver mutations are selected by treatment in clinically aggressive tumors, and indicate a need for longitudinal genomic testing of solid tumors to inform second line cancer treatment.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias Colorrectales/patología , Neoplasias Pulmonares/patología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Mutación , Estadificación de Neoplasias , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Sarcoma/genética , Sarcoma/metabolismo , Sarcoma/patología , Análisis de Secuencia de ADNRESUMEN
Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17ß-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor ß (ERß), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERß. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERß to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.
Asunto(s)
Apoptosis , Endotelio Vascular/metabolismo , Estradiol/metabolismo , Receptor beta de Estrógeno/agonistas , Receptor Notch1/agonistas , Transducción de Señal , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Apoptosis/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/agonistas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
γ-Secretase inhibitors (GSIs) are being actively repurposed as cancer therapeutics based on the premise that inhibition of NOTCH1 signaling in select cancers is therapeutic. Using novel assays to probe effects of GSIs against a broader panel of substrates, we demonstrate that clinical GSIs are pharmacologically distinct. GSIs show differential profiles of inhibition of the various NOTCH substrates, with some enhancing cleavage of other NOTCH substrates at concentrations where NOTCH1 cleavage is inhibited. Several GSIs are also potent inhibitors of select signal peptide peptidase (SPP/SPPL) family members. Extending these findings to mammosphere inhibition assays in triple-negative breast cancer lines, we establish that these GSIs have different functional effects. We also demonstrate that the processive γ-secretase cleavage pattern established for amyloid precursor protein (APP) occurs in multiple substrates and that potentiation of γ-secretase cleavage is attributable to a direct action of low concentrations of GSIs on γ-secretase. Such data definitively demonstrate that the clinical GSIs are not biological equivalents, and provide an important framework to evaluate results from ongoing and completed human trials with these compounds.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , HumanosRESUMEN
BACKGROUND: The lack of early diagnosis, progression markers and effective pharmacological treatment has dramatic unfavourable effects on clinical outcomes in patients with peripheral artery disease (PAD). Addressing these issues will require dissecting the molecular mechanisms underlying this disease. We sought to characterize the Notch signaling and atherosclerosis relevant markers in lesions from femoral arteries of symptomatic PAD patients. METHODS: Plaque material from the common femoral, superficial femoral or popliteal arteries of 20 patients was removed by directional atherectomy. RNA was obtained from 9 out of 20 samples and analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: We detected expression of Notch ligands Delta-like 4 (Dll4) and Jagged1 (Jag1), of Notch target genes Hes1, Hey1, Hey2, HeyL and of markers of plaque inflammation and stability such as vascular cell adhesion molecule 1 (VCAM1), smooth muscle 22 (SM22), cyclooxygenase 2 (COX2), Bcl2, CD68 and miRNAs 21-5p, 125a-5p, 126-5p,146-5p, 155-5p, 424-5p. We found an "inflamed plaque" gene expression profile characterized by high Dll4 associated to medium/high CD68, COX2, VCAM1, Hes1, miR126-5p, miR146a-5p, miR155-5p, miR424-5p and low Jag1, SM22, Bcl2, Hey2, HeyL, miR125a-5p (2/9 patients) and a "stable plaque" profile characterized by high Jag1 associated to medium/high Hey2, HeyL, SM22, Bcl2, miR125a and low Dll4, CD68, COX2, VCAM1, miR126-5p, miR146a-5p, miR155-5p, miR424-5p (3/9 patients). The remaining patients (4/9) showed a plaque profile with intermediate characteristics. CONCLUSIONS: This study reveals the existence of a gene signature associated to Notch activation by specific ligands that could be predictive of PAD progression.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1/metabolismo , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/patología , Placa Aterosclerótica/patología , Transcriptoma , Proteínas Adaptadoras Transductoras de Señales , Anciano , Animales , Proteínas de Unión al Calcio , Colesterol/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Inflamación/patología , Ligandos , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Miocitos del Músculo Liso/metabolismo , Proyectos Piloto , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores Notch/metabolismo , Transducción de Señal/genéticaRESUMEN
Orally bioavailable SERDs may offer greater systemic drug exposure, improved clinical efficacy, and more durable treatment outcome for patients with ER-positive endocrine-resistant breast cancer. We report the design and synthesis of a boronic acid modified fulvestrant (5, ZB716), which binds to ERα competitively (IC50 = 4.1 nM) and effectively downregulates ERα in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. Furthermore, It has superior oral bioavailability (AUC = 2547.1 ng·h/mL) in mice, indicating its promising clinical utility as an oral SERD.
Asunto(s)
Ácidos Borónicos/química , Moduladores Selectivos de los Receptores de Estrógeno/química , Esteroles/química , Administración Oral , Animales , Disponibilidad Biológica , Ácidos Borónicos/síntesis química , Ácidos Borónicos/farmacología , Neoplasias de la Mama , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Resistencia a Antineoplásicos , Receptor alfa de Estrógeno/metabolismo , Femenino , Ratones Endogámicos C57BL , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal , Estereoisomerismo , Esteroles/síntesis química , Esteroles/farmacología , Tamoxifeno/farmacologíaRESUMEN
It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Insuficiencia Cardíaca/sangre , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Receptores Notch/metabolismo , Suero/metabolismo , Transducción de Señal , Anciano , Colágeno/farmacología , Citocinas/sangre , Femenino , Geles/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Masculino , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: Breast cancer is the second leading cause of cancer mortality among women worldwide. The major problem with current treatments is tumor resistance, recurrence, and disease progression. ErbB-2-positive breast tumors are aggressive and frequently become resistant to trastuzumab or lapatinib. We showed previously that Notch-1 is required for trastuzumab resistance in ErbB-2-positive breast cancer. EXPERIMENTAL DESIGN: Here, we sought to elucidate mechanisms by which ErbB-2 attenuates Notch signaling and how this is reversed by trastuzumab or lapatinib. RESULTS: The current study elucidates a novel Notch inhibitory mechanism by which PKCα downstream of ErbB-2 (i) restricts the availability of Jagged-1 at the cell surface to transactivate Notch, (ii) restricts the critical interaction between Jagged-1 and Mindbomb-1, an E3 ligase that is required for Jagged-1 ubiquitinylation and subsequent Notch activation, (iii) reverses trastuzumab resistance in vivo, and (iv) predicts better outcome in women with ErbB-2-positive breast cancer. CONCLUSIONS: The clinical impact of these studies is PKCα is potentially a good prognostic marker for low Notch activity and increased trastuzumab sensitivity in ErbB-2-positive breast cancer. Moreover, women with ErbB-2-positive breast tumors expressing high Notch activation and low PKCα expression could be the best candidates for anti-Notch therapy.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al Calcio/metabolismo , Resistencia a Antineoplásicos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C-alfa/metabolismo , Receptor ErbB-2/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Trastuzumab/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Proteína Jagged-1 , Recurrencia Local de Neoplasia , Pronóstico , Unión Proteica , Transporte de Proteínas , Proteínas Serrate-Jagged , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
p53 and Notch-1 play important roles in breast cancer biology. Notch-1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T-cells but stimulates it in human keratinocytes. Notch co-activator MAML1 binds p53 and functions as a p53 co-activator. We studied the regulation of Notch signaling by p53 in MCF-7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin-3 inhibits Notch-dependent transcriptional activity and Notch target expression in a dose-dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co-immunoprecipitation experiments readily detected a complex containing p53 and Notch-1 in MCF-7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN-MAML1) and stimulated by wild-type MAML1. Standard and quantitative far-Western experiments showed a complex including p53, Notch-1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch-dependent HEY1 promoter and this association is inhibited by DN-MAML1 and stimulated by wild-type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1-dependent fashion, most likely through a p53-MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch-dependent transcription. Our data suggest that p53-null breast cancers may lack this Notch-modulatory mechanism, and that therapeutic strategies that activate wild-type p53 can indirectly cause inhibition of Notch transcriptional activity.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Receptor Notch1/genética , Proteínas Serrate-Jagged , Factores de Transcripción/genética , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Notch/metabolismo , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Línea Celular , Citoplasma , Humanos , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas/química , Receptor Notch4 , Receptores Notch/químicaRESUMEN
Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.
Asunto(s)
Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Receptores Notch/metabolismo , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Embrionario/metabolismo , Transducción de Señal , Línea Celular Tumoral , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/biosíntesis , Factores de Transcripción Paired Box/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch3 , Receptores Notch/genética , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/patología , Rabdomiosarcoma Embrionario/genética , Rabdomiosarcoma Embrionario/patologíaRESUMEN
Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17ß-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Estradiol/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ligandos , Ratones , Neovascularización Fisiológica/efectos de los fármacosRESUMEN
Genome-wide association studies (GWAS) have identified genetic variants associated with an increased risk of developing breast cancer. However, the association of genetic variants and their associated genes with the most aggressive subset of breast cancer, the triple-negative breast cancer (TNBC), remains a central puzzle in molecular epidemiology. The objective of this study was to determine whether genes containing single nucleotide polymorphisms (SNPs) associated with an increased risk of developing breast cancer are connected to and could stratify different subtypes of TNBC. Additionally, we sought to identify molecular pathways and networks involved in TNBC. We performed integrative genomics analysis, combining information from GWAS studies involving over 400,000 cases and over 400,000 controls, with gene expression data derived from 124 breast cancer patients classified as TNBC (at the time of diagnosis) and 142 cancer-free controls. Analysis of GWAS reports produced 500 SNPs mapped to 188 genes. We identified a signature of 159 functionally related SNP-containing genes which were significantly (P <10(-5)) associated with and stratified TNBC. Additionally, we identified 97 genes which were functionally related to, and had similar patterns of expression profiles, SNP-containing genes. Network modeling and pathway prediction revealed multi-gene pathways including p53, NFkB, BRCA, apoptosis, DNA repair, DNA mismatch, and excision repair pathways enriched for SNPs mapped to genes significantly associated with TNBC. The results provide convincing evidence that integrating GWAS information with gene expression data provides a unified and powerful approach for biomarker discovery in TNBC.