RESUMEN
Correction to: Oncogene (2015) 34, 44484459; doi:10.1038/onc.2014.372; published online 24 November 2014. In this article, published online 24 November 2014, the authors have noticed that the latest supplementary information was not used. The corrected supplementary information (Supplementary Materials) appears online together with this corrigendum. The authors would like to apologise for any inconvenience this may cause
RESUMEN
Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.
Asunto(s)
Melanoma/tratamiento farmacológico , Estrés Fisiológico , Animales , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Histona Demetilasas con Dominio de Jumonji/fisiología , Ratones , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores de Factor de Crecimiento Nervioso/fisiología , Proteínas Represoras/fisiología , Transducción de SeñalRESUMEN
Elevated activity of the mitogen-activated protein kinase (MAPK) signaling cascade is found in the majority of human melanomas and is known to regulate proliferation, survival and invasion. Current targeted therapies focus on decreasing the activity of this pathway; however, we do not fully understand how these therapies impact tumor biology, especially given that melanoma is a heterogeneous disease. Using a three-dimensional (3D), collagen-embedded spheroid melanoma model, we observed that MEK and BRAF inhibitors can increase the invasive potential of â¼20% of human melanoma cell lines. The invasive cell lines displayed increased receptor tyrosine kinase (RTK) activity and activation of the Src/FAK/signal transducers and activators of transcription-3 (STAT3) signaling axis, also associated with increased cell-to-cell adhesion and cadherin engagement following MEK inhibition. Targeting various RTKs, Src, FAK and STAT3 with small molecule inhibitors in combination with a MEK inhibitor prevented the invasive phenotype, but only STAT3 inhibition caused cell death in the 3D context. We further show that STAT3 signaling is induced in BRAF-inhibitor-resistant cells. Our findings suggest that MEK and BRAF inhibitors can induce STAT3 signaling, causing potential adverse effects such as increased invasion. We also provide the rationale for the combined targeting of the MAPK pathway along with inhibitors of RTKs, SRC or STAT3 to counteract STAT3-mediated resistance phenotypes.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Neoplasias Cutáneas/metabolismo , Cadherinas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular , Análisis Mutacional de ADN , Resistencia a Antineoplásicos , Humanos , Invasividad Neoplásica , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal , Piel/patologíaRESUMEN
We recently demonstrated that the Adenovirus-5 E1A gene products (E1A), known E2F activators, can block the differentiation of murine preadipocytes and that differentiation suppression occurs at lower levels than required for full neoplastic transformation. Progressively higher levels were accompanied by apoptosis induction. To examine the role of the cellular Ras protooncogene product (Ras) in E1A function, E1A was expressed in C3H10T(1/2) (10T(1/2))-derived preadipocytes rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs (Ras-knockdowns). The results showed that, although even low amounts of E1A could block the differentiation of 10T(1/2) preadipocytes with normal Ras levels, even the highest E1A levels were unable to block the differentiation or induce transformation of Ras-knockdown preadipocytes. Ras downregulation did not affect E2F activation by E1A. Interestingly, our results further demonstrated a dramatic reduction in the levels of the E1A protein itself as differentiation progressed, with a concomitant reduction in E1A's ability to induce apoptosis as a result. These findings suggest for the first time that Ras, although cytoplasmic, is an integral component of the pathway whereby E1A, an oncoprotein believed to have primarily nuclear targets, suppresses differentiation or induces neoplastic conversion of murine preadipocytes.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Adipocitos , Diferenciación Celular , Transformación Celular Neoplásica , Transformación Celular Viral , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Adipocitos/metabolismo , Adipocitos/patología , Animales , Apoptosis , Línea Celular , Regulación Viral de la Expresión Génica , Ratones , TransfecciónRESUMEN
Transfection techniques, such as calcium-phosphate or liposome-mediated gene transfer, are commonly used for the examination of the effect of a gene upon cellular phenotype and biochemical properties. We previously demonstrated that cell to cell adhesion causes a dramatic increase in Stat3 activity. Given that the opportunities for cell to cell adhesion could be altered due to the presence of the DNA-containing complexes, we examined the effect of the calcium-phosphate transfection procedure upon Stat3 activity levels. The results revealed a dramatic increase in Stat3 phosphorylation at the critical tyr705 site and Stat3 activity following calcium-phosphate transfection. This increase was noted even in the absence of DNA and was not due to the mere presence of calcium ions. In contrast, DNA introduction through electroporation or infection with a retroviral vector did not affect Stat3 activity, while cationic lipids such as lipofectamine or Fugene6 had a less pronounced effect than calcium-phosphate transfection. These results indicate that caution is required in the interpretation of results with regard to activity of Stat3 following certain commonly used transient transfection regimens.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Transfección/métodos , Animales , Fosfatos de Calcio , Electroporación , Virus de la Leucemia Murina/genética , Leucemia Experimental/fisiopatología , Lípidos , Liposomas , Ratones , Células 3T3 NIH , Fosforilación , Infecciones por Retroviridae/fisiopatología , Factor de Transcripción STAT3 , Transducción de Señal/fisiología , Infecciones Tumorales por Virus/fisiopatologíaRESUMEN
The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.
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Antígenos Transformadores de Poliomavirus/fisiología , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Dominio Catalítico , Línea Celular , Regulación hacia Abajo , Activación Enzimática , Fibroblastos/metabolismo , Genes ras/genética , Humanos , Insectos , Ratones , Fenotipo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Transfección , Proteínas ras/metabolismoAsunto(s)
Transformación Celular Neoplásica , Transformación Celular Viral , Virus 40 de los Simios/patogenicidad , Virología/métodos , Células 3T3 , Agar , Animales , Antígenos Transformadores de Poliomavirus/genética , Adhesión Celular , Comunicación Celular , Recuento de Células , División Celular , Línea Celular , Medios de Cultivo , Efecto Citopatogénico Viral , Metilcelulosa , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Polihidroxietil Metacrilato , Ratas , Virus 40 de los Simios/genéticaRESUMEN
Activation of the Ras pathway is central to mitogenesis by a variety of growth factors such as the epidermal growth factor, platelet-derived growth factor, or hepatocyte growth factor. Ras activation requires the function of adaptors such as growth factor receptor-binding protein 2, which can bind either directly or indirectly through Src homology 2 domains to the activated receptor. To examine the role of the Src homology 2 domain of growth factor receptor-binding protein 2 in the mitogenic response triggered by these growth factors, we introduced a peptide (PVPE-phosphono-methylphenylalanine-INQS) that can selectively bind this domain into mouse, rat, or human cells growing on conductive indium-tin oxide-coated glass by in situ electroporation. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, extracellular signal-regulated kinase (ERK) 1/2, by probing with antibodies specific for its activated form. Electrodes and slides were configured to provide nonelectroporated control cells side by side with the electroporated ones, both growing on the same type of indium-tin oxide-coated glass surface. The data demonstrate that the peptide can cause a dramatic inhibition of epidermal growth factor or platelet-derived growth factor-mediated ERK1/2 activation and DNA synthesis in vivo, compared with its control phenylalanine-containing counterpart. In contrast, the peptide had a very limited effect on hepatocyte growth factor-triggered ERK1/2 activation and DNA synthesis. These results demonstrate the potential of the in situ electroporation approach described here in the study of the coupling of activated receptor tyrosine kinases to the ERK1/2 cascade.
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Proteínas Adaptadoras Transductoras de Señales , Electroporación/instrumentación , Electroporación/métodos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Péptidos/farmacología , Células 3T3 , Animales , Western Blotting , División Celular , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Flavonoides/farmacología , Proteína Adaptadora GRB2 , Humanos , Inmunohistoquímica , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Tiempo , Dominios Homologos srcRESUMEN
Exercise testing (ET) and adenosine testing (AT) coupled with ECG were applied in 25 patients with old myocardial infarction. ET was positive in 8 (32%) and AT in 10 (40%) patients, the sensitivity of the latter being slightly superior to the former but inferior to the sensitivity reported in the literature for AT-echocardiography. One should also notice the positivity of AT-ECG in 6 (24%) patients, with negative or inconclusive ET, and the double product (DP) at ischemic threshold which was twice as low for AT-ECG as for ET-ECG, proving that AT induced ischemia by decreasing O2 myocardial input. It is concluded that AT-ECG is useful in myocardial ischemia detection in association with ET or in patients who cannot perform ET for different reasons.
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Adenosina , Prueba de Esfuerzo , Infarto del Miocardio/diagnóstico , Adenosina/administración & dosificación , Adulto , Electrocardiografía/efectos de los fármacos , Electrocardiografía/estadística & datos numéricos , Prueba de Esfuerzo/métodos , Prueba de Esfuerzo/estadística & datos numéricos , Femenino , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The possibility of a depressed chronotropic response to exercise in old people as an early signs of sinus node disfunction, was studied in 118 subjects over 60 years (group A) and 25 subjects under 60 years (group B), subjected to a maximal, symptom limited, exercise test (ET) using a cycloergometer. There were no significant statistical differences between the two groups as regards the heart rate (HR) at rest, increase of HR at each effort level, and mean chronotropic impairment (Chl). The mean values were the same when the subjects over 65 years were considered separately (group C). But, when the chronotropic response to ET was studied individually, it was depressed (HR increment less than 15 beats/min at each level) in 22.3% of subjects in group A and only in 4% in group B. The depression was important (increment less than 10/min) in 15% of the subjects in group A and in none of those in group B. The results suggested that about 1/4 of the old people have depressed chronotropic response to exercise attributed by us to a subclinical sinus node disfunction.