RESUMEN
The use of androgen receptor (AR) inhibitors in prostate cancer gives rise to increased cellular lineage plasticity resulting in resistance to AR-targeted therapies. In this study, we examined the chromatin landscape of AR-positive prostate cancer cells post-exposure to the AR inhibitor enzalutamide. We identified a novel regulator of cell plasticity, the homeobox transcription factor SIX2, whose motif is enriched in accessible chromatin regions after treatment. Depletion of SIX2 in androgen-independent PC-3 prostate cancer cells induced a switch from a stem-like to an epithelial state, resulting in reduced cancer-related properties such as proliferation, colony formation, and metastasis both in vitro and in vivo. These effects were mediated through the downregulation of the Wnt/ß-catenin signalling pathway and subsequent reduction of nuclear ß-catenin. Collectively, our findings provide compelling evidence that the depletion of SIX2 may represent a promising strategy for overcoming the cell plasticity mechanisms driving antiandrogen resistance in prostate cancer.
Asunto(s)
Benzamidas , Plasticidad de la Célula , Proteínas de Homeodominio , Nitrilos , Feniltiohidantoína , Neoplasias de la Próstata , Receptores Androgénicos , Vía de Señalización Wnt , beta Catenina , Animales , Humanos , Masculino , Ratones , Benzamidas/farmacología , beta Catenina/metabolismo , beta Catenina/genética , Línea Celular Tumoral , Plasticidad de la Célula/genética , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Nitrilos/farmacología , Células PC-3 , Feniltiohidantoína/farmacología , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The growth factor Neuregulin-1 (NRG-1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG-1/EBBB4-stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG-1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG-1-induced cardiomyocyte hypertrophy. Dynamin-2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin-2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG-1-induced hyperplastic myocardial growth, and chemical inhibition of the Nrg-1/Erbb4 pathway or Dynamin-2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9-mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG-1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG-1/ERBB4/STAT5b pathway in myocardial growth.
Asunto(s)
Dinamina II , Neurregulina-1 , Ratones , Humanos , Animales , Dinamina II/metabolismo , Neurregulina-1/genética , Neurregulina-1/metabolismo , Neurregulina-1/farmacología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Pez Cebra/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , HipertrofiaRESUMEN
Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Comunicación Celular/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Uniones Intercelulares/metabolismo , Uniones Intercelulares/fisiología , Proteínas de la Membrana/metabolismo , Morfogénesis/fisiología , Pez Cebra/fisiologíaRESUMEN
Cancer is a profound medical concern and better treatments are needed for cancer patients. Therefore, new cancer targets are constantly being studied. These targets need not only be relevant for cancer progression, but their modulation needs to be tolerated reasonably well by the host. Caldesmon is one of these proposed novel targets for cancer therapy. Therefore, we analyzed effects of caldesmon mutations in normal development using genetically modified zebrafish embryos. We analyzed mutations in both zebrafish caldesmon genes, cald1a and cald1b and analyzed effects of either mutation alone or as in combination in double homozygous embryos using molecular, morphological and functional analyses. The effects of caldesmon mutations were mild and the gross development of zebrafish embryos was normal. The caldesmon mutant embryos had, however, alterations in response to light-stimulus in behavioural assays. Taken together, the effects of caldesmon mutations in the development of zebrafish embryos were reasonably well tolerated and did not indicate significant concerns for caldesmon being a potential target for cancer therapy.
Asunto(s)
Proteínas de Unión a Calmodulina , Pez Cebra , Animales , Pez Cebra/metabolismo , Proteínas de Unión a Calmodulina/genética , Mutación , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.
Asunto(s)
Proliferación Celular/genética , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1/genética , Células Epiteliales Tiroideas/patología , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Fase G1/efectos de los fármacos , Fase G1/genética , Humanos , Indoles/administración & dosificación , Masculino , Persona de Mediana Edad , Nanopartículas/administración & dosificación , Proteína ORAI1/genética , Polímeros/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Células Epiteliales Tiroideas/efectos de los fármacos , Glándula Tiroides/efectos de los fármacos , Neoplasias de la Tiroides/tratamiento farmacológico , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Adulto Joven , Pez CebraRESUMEN
ß1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes, including cancer cell invasion. Here, we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with ß1-integrin, and its role in recycling of active but not inactive ß1-integrin receptors. Silencing of GGA2 limits active ß1-integrin levels in focal adhesions and decreases cancer cell migration and invasion, which is in agreement with its ability to regulate the dynamics of active integrins. By using the proximity-dependent biotin identification (BioID) method, we identified two RAB family small GTPases, i.e. RAB13 and RAB10, as novel interactors of GGA2. Functionally, RAB13 silencing triggers the intracellular accumulation of active ß1-integrin, and reduces integrin activity in focal adhesions and cell migration similarly to GGA2 depletion, indicating that both facilitate active ß1-integrin recycling to the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Integrina beta1/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Animales Modificados Genéticamente , Adhesión Celular/fisiología , Línea Celular Tumoral , Humanos , Invasividad Neoplásica/patología , Trasplante de Neoplasias , Interferencia de ARN , ARN Interferente Pequeño/genética , Trasplante Heterólogo , Pez Cebra , Proteínas de Unión al GTP rab/genéticaRESUMEN
BACKGROUND: To tackle the missing heritability of sporadic heart failure, we screened for novel heart failure-associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo. METHODS AND RESULTS: Heart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/- cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wildtype genotype or overexpression of WT TRIM55, respectively. CONCLUSIONS: A previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.
Asunto(s)
Exones/genética , Variación Genética , Insuficiencia Cardíaca/genética , Proteínas de Motivos Tripartitos/genética , Actinina/metabolismo , Animales , Secuencia de Bases , Calcio/metabolismo , Cardiomegalia/complicaciones , Cardiomegalia/genética , Cardiomegalia/patología , Ciclo Celular , Línea Celular , Supervivencia Celular , Cromosomas Humanos Par 8/genética , Estudios de Cohortes , Embrión no Mamífero/metabolismo , Finlandia , Regulación de la Expresión Génica , Insuficiencia Cardíaca/fisiopatología , Humanos , Contracción Miocárdica/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteína Sequestosoma-1/metabolismo , Factor de Respuesta Sérica/metabolismo , Estrés Fisiológico/genética , Pez Cebra/embriologíaRESUMEN
Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites, yet only a few of these mutations have been functionally tested. Here, we describe an unbiased platform for the functional characterization of thousands of variants of a single receptor tyrosine kinase (RTK) gene in a single assay. Our in vitroscreen for activating mutations (iSCREAM) platform enabled rapid analysis of mutations conferring gain-of-function RTK activity promoting clonal growth. The screening strategy included a somatic model of cancer evolution and utilized a library of 7,216 randomly mutated epidermal growth factor receptor (EGFR) single-nucleotide variants that were tested in murine lymphoid Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for growth, but this dependence can be compensated by ectopic EGFR overexpression, enabling selection for gain-of-function EGFR mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel activating variant that structurally stabilized the EGFR kinase dimer interface and conferred sensitivity to kinase inhibition by afatinib. As proof of concept for our approach, we recapitulated clinical observations and identified the EGFR L858R as the major enriched EGFR variant. Altogether, iSCREAM enabled robust enrichment of 21 variants from a total of 7,216 EGFR mutations. These findings indicate the power of this screening platform for unbiased identification of activating RTK variants that are enriched under selection pressure in a model of cancer heterogeneity and evolution.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias Pulmonares/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Animales , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Biblioteca de Genes , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , FosforilaciónRESUMEN
BACKGROUND: Receptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart. METHODS: RTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass, was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays. RESULTS: In addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation. CONCLUSIONS: Several novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.
Asunto(s)
Cardiomiopatías/enzimología , Infarto del Miocardio/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocitos Cardíacos/enzimología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Animales , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/genética , Cardiomiopatías/patología , Estudios de Casos y Controles , Línea Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/antagonistas & inhibidores , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Transducción de Señal , Sus scrofaRESUMEN
Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Receptor ErbB-4/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Endocitosis , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Lactancia/genética , Lactancia/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/metabolismo , Ratones , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Embarazo , Transducción de SeñalRESUMEN
Current treatments for anxiety and depression show limited efficacy in many patients, indicating the need for further research into the underlying mechanisms. JNK1 has been shown to regulate anxiety- and depressive-like behaviours in mice, however the effectors downstream of JNK1 are not known. Here we compare the phosphoproteomes from wild-type and Jnk1-/- mouse brains and identify JNK1-regulated signalling hubs. We next employ a zebrafish (Danio rerio) larvae behavioural assay to identify an antidepressant- and anxiolytic-like (AA) phenotype based on 2759 measured stereotypic responses to clinically proven antidepressant and anxiolytic (AA) drugs. Employing machine learning, we classify an AA phenotype from extracted features measured during and after a startle battery in fish exposed to AA drugs. Using this classifier, we demonstrate that structurally independent JNK inhibitors replicate the AA phenotype with high accuracy, consistent with findings in mice. Furthermore, pharmacological targeting of JNK1-regulated signalling hubs identifies AKT, GSK-3, 14-3-3 ζ/ε and PKCε as downstream hubs that phenocopy clinically proven AA drugs. This study identifies AKT and related signalling molecules as mediators of JNK1-regulated antidepressant- and anxiolytic-like behaviours. Moreover, the assay shows promise for early phase screening of compounds with anti-stress-axis properties and for mode of action analysis.
Asunto(s)
Ansiolíticos , Ansiedad , Conducta Animal , Proteína Quinasa 8 Activada por Mitógenos , Transducción de Señal , Pez Cebra , Animales , Ratones , Ansiolíticos/farmacología , Antidepresivos/farmacología , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Larva/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/genética , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.
Asunto(s)
Hemangioma Cavernoso del Sistema Nervioso Central , Células Endoteliales de la Vena Umbilical Humana , Humanos , Animales , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales/metabolismo , Adhesiones Focales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Fibras de Estrés/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Unión Proteica , Ratones , Núcleo Celular/metabolismo , TalinaRESUMEN
Despite clinical benefits of tyrosine kinase inhibitors (TKIs) in cancer, most tumors can reactivate proliferation under TKI therapy. Here we present transcriptional profiling of HER2+ breast cancer cells transitioning from dormant drug tolerant cells to re-proliferating cells under continuous HER2 inhibitor (HER2i) therapy. Focusing on phosphatases, expression of dual-specificity phosphatase DUSP6 was found inhibited in dormant cells, but strongly induced upon regrowth. DUSP6 expression also selectively associated with poor patient survival in HER2+ breast cancers. DUSP6 overexpression conferred apoptosis resistance, whereas its pharmacological blockade prevented therapy tolerance development under HER2i therapy. DUSP6 targeting also synergized with clinically used HER2i combination therapies. Mechanistically DUSP6 is a positive regulator of HER3 expression, and its impact on HER2i tolerance was mediated by neuregulin-HER3 axis. In vivo, genetic targeting of DUSP6 reduced tumor growth in brain metastasis model, whereas its pharmacological targeting induced synthetic lethal therapeutic effect in combination with HER2i. Collectively this work demonstrates that DUSP6 drives escape from HER2i-induced dormancy, and that DUSP6 is a druggable target to overcome HER3-driven TKI resistance.
Asunto(s)
Neoplasias de la Mama , Fosfatasa 6 de Especificidad Dual , Receptor ErbB-2 , Receptor ErbB-3 , Fosfatasa 6 de Especificidad Dual/metabolismo , Fosfatasa 6 de Especificidad Dual/genética , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Femenino , Receptor ErbB-2/metabolismo , Animales , Receptor ErbB-3/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/antagonistas & inhibidores , Línea Celular Tumoral , Ratones , Resistencia a Antineoplásicos/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
BACKGROUND: Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the "metastasis-suppressor" effect of HERC5, with a focus on mitochondrial metabolism pathways. METHODS: We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. RESULTS: Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. CONCLUSIONS: Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Pez Cebra , Regulación hacia Abajo , Ratones Desnudos , Proteómica , Metabolismo Energético , Proliferación Celular , Línea Celular Tumoral , Movimiento Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.
Asunto(s)
Núcleo Celular/metabolismo , Receptores ErbB/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteolisis , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Receptores ErbB/genética , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptor ErbB-4 , Receptores de Cinasa C Activada , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
OBJECTIVE: ErbB4 is a member of the ErbB subfamily of receptor tyrosine kinases with a poorly understood biological role in ovarian cancer. Here, we have addressed the expression, subcellular localization, and prognostic relevance of ErbB4 and its alternatively spliced isoforms in serous ovarian adenocarcinoma. METHODS: A tissue microarray including 482 samples was analyzed by immunohistochemistry, and a series of 198 samples by isoform-specific real-time RT-PCR. The data were statistically analyzed for associations with clinicopathological markers and survival. The functional effect of expressing the relevant ErbB4 isoforms in ovarian cancer cells was addressed by measuring colony formation in soft agar. RESULTS: While ErbB4 immunoreactivity was present in 90% of the samples, total ErbB4 protein expression was not significantly associated with prognostic markers. However, real-time RT-PCR analysis of serous ovarian cancer samples indicated the presence of two alternatively spliced cytoplasmic isoforms of ERBB4, CYT-1 and CYT-2, previously demonstrated to mediate significantly different cellular activities. Expression of CYT-1, but not of CYT-2, was significantly associated with tumor grade (P=0.014) and poor overall survival (P=0.0028). CYT-1 expression was also an independent prognostic factor (P=0.021) in multivariate analysis of survival. Consistent with a biological effect specific for the one isoform, overexpression of ErbB4 CYT-1, but not of ErbB4 CYT-2, increased anchorage-independent growth of ovarian adenocarcinoma cells in vitro. CONCLUSIONS: These results suggest that expression of a specific ErbB4 isoform, CYT-1, is associated with poor survival and enhanced growth in serous ovarian cancer.
Asunto(s)
Cistadenocarcinoma Seroso/enzimología , Receptores ErbB/análisis , Neoplasias Ováricas/enzimología , Adulto , Anciano , Proliferación Celular , Cistadenocarcinoma Seroso/mortalidad , Cistadenocarcinoma Seroso/patología , Receptores ErbB/fisiología , Femenino , Humanos , Inmunohistoquímica , Isoenzimas/análisis , Persona de Mediana Edad , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Pronóstico , Receptor ErbB-4 , Análisis de Matrices TisularesRESUMEN
Power outages can happen anywhere and anytime for various reasons. This threat affects also scientific work of biologists. Especially problematic area is aquatic animal husbandry, where life support of the animals is dependent on continuous electricity supply and years of scientific work may depend on the well-being of these animal stocks. Therefore, tools to estimate and control these risks are needed. In this study, I have used modeling to estimate aquarium water temperature changes during power outages and constructed simplified models for zebrafish aquaria. A calculation worksheet is also provided to help to model kinetics of water temperature changes in zebrafish facilities.
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Vivienda , Pez Cebra , Animales , Temperatura , Crianza de Animales Domésticos , AguaRESUMEN
Impaired wound healing is associated with aging and has significant effects on human health on an individual level, but also on the whole health-care sector. Deficient angiogenesis appears to be involved in the process, but the underlying biology is still poorly understood. This is at least partially being explained by complexity and costs in using mammalian aging models. To understand aging-related vascular biology of impaired wound healing, we used zebrafish and turquoise killifish fin regeneration models. The regeneration of caudal fin after resection was significantly reduced in old individuals in both species. Age-related changes in angiogenesis, vascular density and expression levels of angiogenesis biomarker VEGF-A were observed. Furthermore, the anti-angiogenic drug vascular endothelial growth factor receptor blocking inhibitor SU5416 reduced regeneration, indicating a key role for angiogenesis in the regeneration of aging caudal fin despite aging-related changes in vasculature. Taken together, our data indicate that these fish fin regeneration models are suitable for studying aging-related decline in wound healing and associated alterations in aging vasculature.
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Fundulidae , Pez Cebra , Animales , Humanos , Anciano , Pez Cebra/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Pez Cebra , Cicatrización de Heridas , Mamíferos/metabolismoRESUMEN
Lethal prostate cancer (PCa) is characterized by the presence of metastases and development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. Here, we report that in vitro downregulation of l-CaD promotes epithelial phenotype and reduces spheroid growth in 3D, which is reflected in vivo in reduced formation of metastases in zebrafish PCa xenografts. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition (EMT) transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets, and GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation.
RESUMEN
While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.