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Emerging evidence indicates the presence of vascular abnormalities and ischemia in biliary atresia (BA), although specific mechanisms remain undefined. This study examined both human and experimental BA. Structural and hemodynamic features of hepatic arteries were investigated by Doppler ultrasound, indocyanine green angiography, microscopic histology, and invasive arterial pressure measurement. Opal multiplex immunohistochemistry, western blot, and RT-PCR were applied to assess Notch3 expression and the phenotype of hepatic arterial smooth muscle cells (HASMCs). We established animal models of Notch3 inhibition, overexpression, and knockout to evaluate the differences in overall survival, hepatic artery morphology, peribiliary hypoxia, and HASMC phenotype. Hypertrophic hepatic arteriopathy was evidenced by an increased wall-to-lumen ratio and clinically manifested as hepatic arterial hypertension, decreased hepatic artery perfusion, and formation of hepatic subcapsular vascular plexuses (HSVPs). We observed a correlation between overactivation of Notch3 and phenotypic disruption of HASMCs with the exacerbation of peribiliary hypoxia. Notch3 signaling mediated the phenotype alteration of HASMCs, resulting in arterial wall thickening and impaired oxygen supply in the portal microenvironment. Inhibition of Notch3/Hey1 ameliorates portal hypoxia by restoring the balance of contractile/synthetic HASMCs, thereby preventing hypertrophic arteriopathy in BA.
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Atresia Biliar , Receptor Notch3 , Receptor Notch3/metabolismo , Receptor Notch3/antagonistas & inhibidores , Animales , Atresia Biliar/patología , Atresia Biliar/metabolismo , Ratones , Humanos , Masculino , Hipoxia/metabolismo , Femenino , Arteria Hepática/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: The leading cause of mortality in patients with Marfan syndrome (MFS) is thoracic aortic aneurysm and dissection. Notch signaling is essential for vessel morphogenesis and function. However, the role of Notch signaling in aortic pathology and aortic smooth muscle cell (SMC) differentiation in Marfan syndrome (MFS) is not completely understood. METHODS: RNA-sequencing on ascending aortic tissue from a mouse model of MFS, Fbn1mgR/mgR , and wild-type controls was performed. Notch 3 expression and activation in aortic tissue were confirmed with real-time RT-PCR, immunohistochemistry, and Western blot. Fbn1mgR/mgR and wild-type mice were treated with a γ-secretase inhibitor, DAPT, to block Notch activation. Aortic aneurysms and rupture were evaluated with connective tissue staining, ultrasound, and life table analysis. RESULTS: The murine RNA-sequencing data were validated with mouse and human MFS aortic tissue, demonstrating elevated Notch3 activation in MFS. Data further revealed that upregulation and activation of Notch3 were concomitant with increased expression of SMC contractile markers. Inhibiting Notch3 activation with DAPT attenuated aortic enlargement and improved survival of Fbn1mgR/mgR mice. DAPT treatment reduced elastin fiber fragmentation in the aorta and reversed the differentiation of SMCs. CONCLUSIONS: Our data demonstrated that matrix abnormalities in the aorta of MFS are associated with increased Notch3 activation. Enhanced Notch3 activation in MFS contributed to aortic aneurysm formation in MFS. This might be mediated by inducing a contractile phenotypic change of SMC. Our results suggest that inhibiting Notch3 activation may provide a strategy to prevent and treat aortic aneurysms in MFS.
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Aorta/patología , Aneurisma de la Aorta/metabolismo , Síndrome de Marfan/metabolismo , Miocitos del Músculo Liso/fisiología , Receptor Notch3/metabolismo , Animales , Aneurisma de la Aorta/genética , Diaminas/administración & dosificación , Diaminas/farmacología , Modelos Animales de Enfermedad , Elastina/metabolismo , Fibrilina-1/genética , Fibrilina-1/metabolismo , Humanos , Síndrome de Marfan/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Terapia Molecular Dirigida , Receptor Notch3/antagonistas & inhibidores , Tiazoles/administración & dosificación , Tiazoles/farmacologíaRESUMEN
BACKGROUND: The transformation of hepatic stellate cells (HSCs) into collagen-producing myofibroblasts is a key event in hepatic fibrogenesis. Recent studies have shown that microRNAs (miRNAs) play a critical role in the transformation of HSCs. However, the function of miR-489-3p in liver fibrosis remains unclear. METHODS: Here, we detected the levels of miR-489-3p and jagged canonical Notch ligand 1 (JAG1) in liver fibrosis by using CCl4-treated rats as an in vivo model and transforming growth factor-beta 1 (TGF-ß1)-treated HSC cell lines LX-2 and HSC-T6 as in vitro models. The expression of profibrotic markers was affected by transfecting LX-2 cells with either miR-489-3p mimic or si-JAG1. A dual-luciferase reporter assay was carried out to study the interaction of JAG1 with miR-489-3p. RESULTS: We found that miR-489-3p was remarkably decreased while JAG1 was increased in liver fibrosis models both in vivo and in vitro. Overexpression of miR-489-3p reduced the expression of profibrotic markers and the activation of LX-2 cells induced by TGF-ß1. Moreover, miR-489-3p decreased the expression of jagged canonical Notch ligand 1 (JAG1) in LX-2 cells by interacting with its 3'-UTR. As JAG1 is a Notch ligand, decreased JAG1 by miR-489-3p inhibited the Notch signaling pathway. Moreover, the downregulation of JAG1 inhibited the expression of fibrotic markers. CONCLUSION: Our results indicate that miR-489-3p can inhibit HSC activation by inhibiting the JAG1/Notch3 signaling pathway.
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Células Estrelladas Hepáticas/metabolismo , Proteína Jagged-1/biosíntesis , Cirrosis Hepática/metabolismo , MicroARNs/biosíntesis , Receptor Notch3/biosíntesis , Transducción de Señal/fisiología , Animales , Línea Celular , Células Estrelladas Hepáticas/patología , Humanos , Proteína Jagged-1/antagonistas & inhibidores , Cirrosis Hepática/patología , Masculino , Ratas , Ratas Sprague-Dawley , Receptor Notch3/antagonistas & inhibidoresRESUMEN
BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an orphan disease clinically characterized by migraine, recurrent strokes, and dementia. Currently, there are no disease-modifying therapies, and it is difficult to prevent cerebral ischemic events in CADASIL patients by conventional antithrombotic medication. We hypothesized that an antimigraine agent, lomerizine hydrochloride, may prevent strokes in CADASIL patients, based on its effect on increasing cerebral blood flow. SUBJECTS AND METHODS: This was an open-labeled clinical trial in which 30 adult CADASIL patients received lomerizine at 10 mg/d. Numbers of symptomatic strokes during the 2 years after the start of lomerizine administration were compared with those in the 2 years before its initiation. The effect of lomerizine on preventing strokes was evaluated based on the incidence rate ratio (IR) calculated with the Mantel-Haenszel method. RESULTS: When including all 30 patients (analysis 1), the IR was less than 1 (0.46; 95% confidence interval [CI], 0.19-1.12) but did not reach significance. To evaluate the effect of lomerizine on secondary prevention, subgroups of 15 patients with stroke episodes occurring any time before lomerizine administration (analysis 2) and 10 patients with stroke episodes during the 2 years before lomerizine administration (analysis 3) were analyzed. The IR values were 0.33 (95% CI, 0.12-0.94) in analysis 2 and 0.17 (95% CI, 0.04-0.67) in analysis 3. CONCLUSIONS: Our results suggest the effect of lomerizine on preventing secondary stroke in CADASIL patients.
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CADASIL/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/prevención & control , Piperazinas/uso terapéutico , Adulto , Anciano , CADASIL/complicaciones , Femenino , Humanos , Incidencia , Accidente Cerebrovascular Isquémico/complicaciones , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Proyectos Piloto , Piperazinas/efectos adversos , Receptor Notch3/antagonistas & inhibidores , Prevención SecundariaRESUMEN
The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.
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Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Receptor Notch3/metabolismo , Transducción de Señal , Membrana Sinovial/patología , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Células Endoteliales/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Receptor Notch3/antagonistas & inhibidores , Receptor Notch3/deficiencia , Receptor Notch3/genética , Antígenos Thy-1/metabolismoRESUMEN
Background PF-06650808 is a novel anti-Notch3 antibody-drug conjugate (ADC) able to deliver an auristatin-based cytotoxic payload to target cells. In this first-in-human, dose-finding, phase I study (NCT02129205), we investigated safety, pharmacokinetics, immunogenicity, and preliminary antitumor activity of single-agent PF-06650808 in 40 patients with advanced breast cancer (BC) and other solid tumors unselected for Notch3 expression. Primary endpoint was dose-limiting toxicity (DLT). PF-06650808 was administered intravenously every 3 weeks at a starting dose of 0.2 mg/kg, escalated up to 6.4 mg/kg following the modified continual reassessment method. An additional dose level, 2.0 mg/kg, was evaluated in patients with advanced, estrogen receptor-positive (ER+) BC. Results The majority of patients had advanced BC (60%) and almost all (90%) had received ≥3 prior lines of anticancer therapy. Treatment with PF-06650808 was generally well tolerated at dose levels ≤2.0 mg/kg with no DLTs. The maximum tolerated dose (MTD) was estimated to be 2.4 mg/kg. The most common treatment-related AEs in all patients were fatigue (40.0%), decreased appetite (37.5%), nausea (35.0%), alopecia (32.5%), abdominal pain (25.0%), pruritus (25.0%), and vomiting (25.0%). Five patients achieved a partial response (PR), including 2 unconfirmed PRs; 4 of the responders had ER+/PR+/HER2- BC. Sixteen (51.6%) patients achieved stable disease, including 8 (57.1%) of 14 patients with ER+ BC. Tumor samples from all responders tested positive for NOTCH3 expression in a retrospective, exploratory analysis. Conclusions The anti-Notch3 ADC PF-06650808 has demonstrated a manageable safety profile and early signs of antitumor activity in patients with advanced BC.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Inmunoconjugados/química , Neoplasias/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Receptor Notch3/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos Inmunológicos/farmacocinética , Neoplasias de la Mama/secundario , Femenino , Estudios de Seguimiento , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias/patología , Oligopéptidos/farmacocinética , Pronóstico , Receptor Notch3/inmunología , Estudios Retrospectivos , Distribución Tisular , Adulto JovenRESUMEN
PURPOSE: Notch signaling dysregulation is implicated in the development of pancreatic adenocarcinoma (PDAC). Tarextumab is a fully human IgG2 antibody that inhibits Notch2/3 receptors. PATIENTS AND METHODS: Aphase 2, randomized, placebo-controlled, multicenter trial evaluated the activity of tarextumab in combination with nab-paclitaxel and gemcitabine in patients with metastatic PDAC. Patients were stratified based on ECOG performance score and Ca 19-9 level and randomized 1:1 to nab-paclitaxel, gemcitabine with either tarextumab or placebo. Based on preclinical and phase Ib results suggesting a positive correlation between Notch3 gene expression and tarextumab anti-tumor activity, patients were also divided into subgroups of low, intermediate, and high Notch3 gene expression. Primary endpoint was overall survival (OS) in all and in patients with the three Notch3 gene expression subgroups (≥25th, ≥50% and ≥75% percentiles); secondary end points included progression-free survival (PFS), 12-month OS, overall response rate (ORR), and safety and biomarker investigation. RESULTS: Median OS was 6.4 months in the tarextumab group vs 7.9 months in the placebo group (HR = 1.34 [95% CI = 0.95, 1.89], P = .0985). No difference observed in OS in the Notch3 gene expression subgroups. PFS in the tarextumab-treated group (3.7 months) was significantly shorter compared with the placebo group (5.5 months) (hazard ratio was 1.43 [95% CI = 1.01, 2.01]; P = .04). Grade 3 diarrhea and thrombocytopenia were more common in the tarextumab group. CONCLUSIONS: The addition of tarextumab to nab-paclitaxel and gemcitabine did not improve OS, PFS, or ORR in first-line metastatic PDAC, and PFS was specifically statistically worse in the tarextumab-treated patients. CLINICAL TRIAL REGISTRY NO: NCT01647828.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Albúminas/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Biomarcadores , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Manejo de la Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Pronóstico , Receptor Notch2/antagonistas & inhibidores , Receptor Notch3/antagonistas & inhibidores , Resultado del Tratamiento , GemcitabinaRESUMEN
BACKGROUND: Sorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results. METHODS: 2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers. RESULTS: Using a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo. CONCLUSION: We have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.
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Alanina/análogos & derivados , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Receptor Notch3/antagonistas & inhibidores , Triazinas/farmacología , Alanina/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Electroforesis en Gel de Poliacrilamida , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/terapia , Células MCF-7 , Terapia Molecular Dirigida , Proteómica , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Wistar , Receptor Notch3/deficiencia , Receptor Notch3/genética , Electroforesis Bidimensional Diferencial en GelRESUMEN
Purpose This Phase I trial evaluated the maximum tolerated dose, safety, pharmacokinetics, pharmacodynamics and preliminary efficacy of tarextumab (OMP-5948), a novel cross-reactive antibody which binds and selectively inhibits signaling via both Notch2 and Notch3, in adult patients with advanced malignancies. Methods Standard 3 + 3 design with tarextumab 0.5, 1, 2.5, or 5 mg/kg weekly, or 5, 7.5, or 10 mg/kg every other week, or 7.5 mg every 3 weeks. Dose-limiting toxicities (DLT) were assessed during the first 28 days. Results Forty-two patients received tarextumab (21 weekly, 15 every other week, 6 every three weeks). 2/6 subjects at the 5 mg/kg weekly dose, 2/3 at 10 mg/kg every other week, and 0/6 at 7.5 mg/kg every three weeks had a DLT. The maximum tolerated dose (MTD) was 2.5 mg/kg weekly, and 7.5 mg/kg on the every other and every three week schedules. Gastrointestinal (GI) toxicity was the most common adverse event with diarrhea (81%), fatigue (48%), nausea (45%), anorexia (38%), and vomiting (38%) and abdominal pain and constipation (24% each). Biomarker analysis showed regulation of stem cell and Notch gene signaling. Conclusion Tarextumab was generally well-tolerated at doses <2.5 mg weekly and 7.5 mg/kg every other and every third week. Diarrhea was dose-limiting above these levels, but relatively easily managed at lower doses. Inhibition of Notch pathway signaling was demonstrated at these doses. ClinicalTrials.gov Identifier: NCT01277146.
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Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias/tratamiento farmacológico , Receptor Notch2/antagonistas & inhibidores , Receptor Notch3/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Anorexia/inducido químicamente , Anticuerpos/sangre , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Antineoplásicos/efectos adversos , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Diarrea/inducido químicamente , Fatiga/inducido químicamente , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Náusea/inducido químicamente , Neoplasias/genética , Neoplasias/metabolismo , Receptor Notch2/genética , Receptor Notch3/genética , Transcriptoma , Vómitos/inducido químicamenteRESUMEN
Notch3 is a receptor of the Notch signaling pathway and plays an important role in regulating selfrenewal, differentiation and apoptosis in cancer cells. Overexpression of Notch3 has been proved to be associated with resistance to gemcitabine (GEM) and poor patient prognosis for various malignant tumors. In the present study, two nonsmall cell lung cancer (NSCLC) cell lines, H1299 and A549, were induced with GEM for two months and then were treated with various concentrations of a Notch signaling blocker, N[N(3,5difluorophenacetyl)Lalanyl]Sphenylglycine tbutyl ester (DAPT), with the goal of reducing expression of Notch intracellular domain 3 (NICD3). Both cell lines were subsequently treated with either DAPT or DAPT combined with GEM and then viability, apoptosis, colony formation and cell count assays were performed. DAPT treatment effectively downregulated the expression of NICD3 in both cell lines. DAPT combined with GEM also significantly reduced the percentage of viable cells in both cell lines, while increasing the percentage of apoptotic cells, compared with GEM alone. In the clonogenicity assays, the combination of DAPT and GEM led to a decrease in clone numbers and significantly greater inhibition of the H1299 and A549 cells compared to treatment with DAPT or GEM alone. Meanwhile, levels of the apoptosisrelated proteins, Bcl2 and Bax, were found to be affected by the various treatments. Thus Notch3 appears to be a promising target for gene therapy and DAPT is able to mediate a strong antitumor effect in NSCLC cells that overexpress Notch3. Further studies of a combined treatment regimen with DAPT and GEM are warranted and may provide greater efficacy and safety in the treatment of NSCLC patients.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Diaminas/farmacología , Receptor Notch3/genética , Tiazoles/farmacología , Células A549 , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Desoxicitidina/efectos adversos , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Receptor Notch3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , GemcitabinaRESUMEN
BACKGROUND: Activated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC. METHODS: The expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student's t-test. RESULTS: Notch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells. CONCLUSIONS: Notch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the PaSC-induced pro-tumorigenic effect. Therefore, Notch3 silencing in PaSCs is a potential novel therapeutic option for patients with PDAC.
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Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Células Estrelladas Pancreáticas/patología , Receptor Notch3/genética , Actinas/genética , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Colágeno Tipo I/genética , Fibronectinas/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Receptor Notch3/antagonistas & inhibidoresRESUMEN
BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) is a pivotal regulator of cell proliferation, survival, and autophagy. Autophagy is increased in adult experimental chronic pulmonary hypertension (PHT), but its contributory role to pulmonary vascular disease remains uncertain and has yet to be explored in the neonatal animal. Notch is a major pro-proliferative pathway activated by mTOR. A direct relationship between autophagy and Notch signaling has not been previously explored. Our aim was to examine changes in mTOR-, Notch-, and autophagy-related pathways and the therapeutic effects of autophagy modulators in experimental chronic neonatal PHT secondary to chronic hypoxia. METHODS: Rat pups were exposed to normoxia or hypoxia (13% O2 ) from postnatal days 1-21, while receiving treatment with temsirolimus (mTOR inhibitor), DAPT (Notch inhibitor), or chloroquine (inhibitor of autophagic flux). RESULTS: Exposure to hypoxia up-regulated autophagy and Notch3 signaling markers in lung, pulmonary artery (PA), and PA-derived smooth muscle cells (SMCs). Temsirolimus prevented chronic PHT and attenuated PA and SMC signaling secondary to hypoxia. These effects were replicated by DAPT. mTOR or Notch inhibition also down-regulated smooth muscle content of platelet-derived growth factor ß-receptor, a known contributor to vascular remodeling. In contrast, chloroquine had no modifying effects on markers of chronic PHT. Knockdown of Beclin-1 in SMCs had no effect on hypoxia-stimulated Notch3 signaling. CONCLUSIONS: mTOR-Notch3 signaling plays a critical role in experimental chronic neonatal PHT. Inhibition of autophagy did not suppress Notch signaling and had no effect on markers of chronic PHT.
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Hipertensión Pulmonar/metabolismo , Receptor Notch3/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Animales Recién Nacidos , Autofagia , Proliferación Celular/efectos de los fármacos , Diaminas/farmacología , Femenino , Hipoxia/metabolismo , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Masculino , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Ratas Sprague-Dawley , Receptor Notch3/antagonistas & inhibidores , Transducción de Señal , Sirolimus/análogos & derivados , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Tiazoles/farmacologíaRESUMEN
BACKGROUND Lung cancers are resistant to conventional chemotherapeutic interventions such as paclitaxel. Notch signaling is crucial in the chemoresistance of lung cancer cells. The Notch inhibitor gamma-secretase inhibitor (GSI) inhibits the Notch signaling pathway. MATERIAL AND METHODS Here, we evaluated how Notch-3 inhibition by GSI can enhance the sensitivity of lung cancer cells to paclitaxel. To study how Notch-3-specific inhibition affects non-small cell lung cancer (NSCLC), we compared the cell viability, apoptosis, and colony formation of A549 and H1299 cells treated with Notch-3 siRNA and GSI. RESULTS The expression levels of Notch-3 or Notch intracellular domain 3 (NICD3) and apoptosis-related proteins were measured and compared between different groups. Notch-3 was significantly overexpressed in both cell lines, and Notch-3 expression was elevated after paclitaxel treatment, indicating activation of the Notch signaling pathway. Inhibition of the Notch signaling pathway by GSI and Notch-3 siRNA reduced cell proliferation and induced apoptosis in A549 and H1299 cells, thereby boosting sensitivity of the cell lines to paclitaxel. Concomitant treatment with paclitaxel and GSI or siRNA downregulated Bcl-2 expression and upregulated Bax expression levels. CONCLUSIONS These results indicate a synergistic effect of Notch-3-specific inhibition and paclitaxel through alteration of the intrinsic apoptosis pathway, which was involved in Notch-3-induced chemoresistance in NSCLC cells, and GSI inhibited Notch-3-induced chemoresistance in a concentration-dependent manner. This approach that combines Notch-3-specific inhibition and paclitaxel would be likely to apply in NSCLC.
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Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Paclitaxel/uso terapéutico , Receptor Notch3/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Paclitaxel/farmacología , Receptor Notch3/metabolismo , Ensayo de Tumor de Célula Madre , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.
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Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , Receptor Notch3/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Diferenciación Celular , Línea Celular Transformada , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Genes Reporteros , Células HEK293 , Semivida , Humanos , Imidazoles/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Naftalenos/farmacología , Cultivo Primario de Células , Próstata/citología , Próstata/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Notch3/antagonistas & inhibidores , Receptor Notch3/metabolismo , Transducción de Señal , Tiazoles/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: The role of the Notch pathway has already been identified as a crucial regulator of bone development. However, the Notch signaling pathway has gone largely unexplored during osseointegration. This study aims to investigate the role of Notch signaling on osteogenic differentiation of rat derived bone marrow mesenchymal stem cells (BMSCs) on sandblasted, large-grit, acid-etched (SLA) treated Ti disks. METHODS: The involved target genes in Notch pathways were identified by in vitro microarray and bioinformatics analyses with or without osteogenic induction. Adhesion, proliferation, and osteogenic related assay were subsequently conducted with target gene shRNA treatment. RESULTS: We found that 11 genes in the Notch signaling pathway were differentially expressed after osteogenic induction on SLA-treated Ti disks, which included up-regulated genes (Notch2, Dll1, Dll3, Ncstn, Ncor2, and Hes5) and down-regulated genes (Notch3, Lfng, Mfng, Jag2 and Maml2). With Notch3 shRNA treatment, the adhesion and proliferation of BMSCs on SLA-treated Ti disks were inhibited. Moreover, the expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), calcium deposition, BMP2 and Runx2 increased significantly compared with that observed in control groups, suggesting that the function of Notch3 was inhibitory in the osteogenic differentiation of BMSCs on SLA-treated titanium. CONCLUSIONS: Inhibition Notch3 can enhance osteogenic differentiation of BMSCs on SLA-treated Ti disks, which potentially provides a gene target for improving osseointegration.
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Células de la Médula Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , Receptor Notch3/antagonistas & inhibidores , Grabado Ácido Dental , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Proteína Morfogenética Ósea 2/metabolismo , Calcificación Fisiológica , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Masculino , Análisis por Micromatrices , Microscopía Electrónica de Rastreo , Osteocalcina/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Propiedades de Superficie , TitanioRESUMEN
Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch3/antagonistas & inhibidores , Receptor Notch3/genética , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Codón , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/inmunología , Femenino , Humanos , Ratones , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Conformación Proteica , Receptor Notch3/química , Receptor Notch3/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acquired resistance to standard chemotherapy causes treatment failure in patients with local advanced and advanced non-small lung cancer (NSCLC). Cancer stem cells (CSCs) are a small subpopulation within cancer that is thought to be resistant to conventional chemotherapy. The Notch pathway is one of the most intensively studied for putative therapeutic targets of CSCs in solid tumors. In our study, suppression of Notch3 decreased colony and sphere formation of stem-like property in lung cancer cells. In addition, Notch3 expression was demonstrated to be upregulated in the patients with chemoresistance and related to poor prognosis of NSCLC patients. Our results also showed that CSC markers ALDH1A1 and CD44 were highly expressed in NSCLC patients with chemoresistance and these two markers were positively correlated with Notch3 expression in lung cancer specimens from TCGA database. Furthermore, the lung cancer cells with drug resistance were shown to be associated with activation of autophagy. All the data support a crucial role of Notch3 in the increase of stem-like property in NSCLC cells that might be associated with upregulation of ALDH1A1 and CD44 and activation of autophagy.
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Aldehído Deshidrogenasa/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores de Hialuranos/biosíntesis , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptor Notch3/antagonistas & inhibidores , Células A549 , Familia de Aldehído Deshidrogenasa 1 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Cisplatino/farmacología , Resistencia a Antineoplásicos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Receptor Notch3/metabolismo , Retinal-DeshidrogenasaRESUMEN
Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.
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Carcinoma de Células Escamosas/genética , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca/genética , Neovascularización Patológica/genética , Receptor Notch3/genética , Adulto , Anciano , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Comunicación Celular , Técnicas de Cocultivo , Femenino , Fibroblastos/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/mortalidad , Neoplasias de la Boca/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Pronóstico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Notch3/antagonistas & inhibidores , Receptor Notch3/metabolismo , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
Notch signaling plays an important role in ovarian cancer chemoresistance, which is responsible for recurrence. Gamma-secretase inhibitor (GSI) is a broad-spectrum Notch inhibitor, but it has serious side effects. The efficacy of Notch3-specific inhibition in paclitaxel-resistant ovarian cancers was assessed in this study, which has not yet been evaluated relative to GSI. To analyze the effect of Notch3-specific inhibition on paclitaxel-resistant ovarian cancers, we compared cell viability, apoptosis, cell migration, angiogenesis, cell cycle, and spheroid formation after treatment with either Notch3 siRNA or GSI in paclitaxel-resistant SKpac cells and parental SKOV3 cells. Expression levels of survival, cell cycle, and apoptosis-related proteins were measured and compared between groups. Notch3 was significantly overexpressed in chemoresistant cancer tissues and cell lines relative to chemosensitive group. In paclitaxel-resistant cancer cells, Notch inhibition significantly reduced viability, migration, and angiogenesis and increased apoptosis, thereby boosting sensitivity to paclitaxel. Spheroid formation was also significantly reduced. Both Notch3 siRNA-treated cells and GSI-treated cells arrested in the G2/M phase of the cell cycle. Proteins of cell survival, cyclin D1 and cyclin D3 were reduced, whereas p21 and p27 were elevated. Both GSI and Notch3 siRNA treatment reduced expression of anti-apoptotic proteins (BCL-W, BCL2, and BCL-XL) and increased expression of pro-apoptotic proteins (Bad, Bak, Bim, Bid, and Bax). These results indicate that Notch3-specific inhibition sensitizes paclitaxel-resistant cancer cells to paclitaxel treatment, with an efficacy comparable to that of GSI. This approach would be likely to avoid the side effects of broad-spectrum GSI treatment. © 2015 Wiley Periodicals, Inc.
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Resistencia a Antineoplásicos/efectos de los fármacos , Oligopéptidos/farmacología , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , ARN Interferente Pequeño/farmacología , Receptor Notch3/genética , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Paclitaxel/farmacología , Receptor Notch3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27(Kip) accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27(Kip) at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27(Kip) accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.