RESUMEN
Immune checkpoint inhibitors (ICIs) have rejuvenated therapeutic approaches in oncology. Although responses tend to be durable, response rates vary in many cancer types. Thus, the identification and validation of predictive biomarkers is a key clinical priority, the answer to which is likely to lie in the tumour microenvironment (TME). A wealth of data demonstrates the huge impact of the TME on ICI response and resistance. However, these data also reveal the complexity of the TME composition including the spatiotemporal interactions between different cell types and their dynamic changes in response to ICIs. Here, we briefly review some of the modalities that sculpt the TME, in particular the metabolic milieu, hypoxia and the role of cancer-associated fibroblasts. We then discuss recent approaches to dissect the TME with a focus on single-cell RNA sequencing, spatial transcriptomics and spatial proteomics. We also discuss some of the clinically relevant findings these multi-modal analyses have yielded.
RESUMEN
The integrity of the vasculature plays an important role in the success of allogeneic organ and haematopoietic stem cell transplantation. Endothelial cells (EC) have previously been shown to be the target of activated cytotoxic T lymphocytes (CTL) resulting in extensive cell lysis. Mesenchymal stromal cells (MSC) are multipotent cells which can be isolated from multiple sites, each demonstrating immunomodulatory capabilities. They are explored herein for their potential to protect EC from CTL-targeted lysis. CD8(+) T cells isolated from human PBMC were stimulated with mitotically inactive cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as HMEC) for 7 days. Target HMEC were cultured in the presence or absence of MSC for 24 h before exposure to activated allogeneic CTL for 4 h. EC were then analysed for cytotoxic lysis by flow cytometry. Culture of HMEC with MSC in the efferent immune phase (24 h before the assay) led to a decrease in HMEC lysis. This lysis was determined to be MHC Class I restricted linked and further analysis suggested that MSC contact is important in abrogation of lysis, as protection is reduced where MSC are separated in transwell experiments. The efficacy of multiple sources of MSC was also confirmed, and the collaborative effect of MSC and the endothelium protective drug defibrotide were determined, with defibrotide enhancing the protection provided by MSC. These results support the use of MSC as an adjuvant cellular therapeutic in transplant medicine, alone or in conjunction with EC protective agents such as defibrotide.
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Citotoxicidad Inmunológica , Células Endoteliales/inmunología , Células Madre Mesenquimatosas/inmunología , Factores Protectores , Linfocitos T Citotóxicos/inmunología , Comunicación Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Polidesoxirribonucleótidos/farmacología , Cultivo Primario de Células , Sustancias Protectoras/farmacología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacosRESUMEN
A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficiently inhibiting apoptosis. Here, we show that the interaction of A-Raf with the MST2 pathway is regulated by subcellular compartmentalization. Although in proliferating normal cells and tumor cells A-Raf localizes to the mitochondria, differentiated non-carcinogenic cells of head and neck epithelia, which express A-Raf at the plasma membrane. The constitutive or induced re-localization of A-Raf to the plasma membrane compromises its ability to efficiently sequester and inactivate MST2, thus rendering cells susceptible to apoptosis. Physiologically, A-Raf re-localizes to the plasma membrane upon epithelial differentiation in vivo. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Downregulation of KSR2 during mammary epithelial cell differentiation or siRNA-mediated knockdown re-localizes A-Raf to the plasma membrane causing the release of MST2. By using the MCF7 cell differentiation system, we could demonstrate that overexpression of A-Raf in MCF7 cells, which induces differentiation. Our findings offer a new paradigm to understand how differential localization of Raf complexes affects diverse signaling functions in normal cells and carcinomas.
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Apoptosis , Diferenciación Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas A-raf/metabolismo , Caspasa 8/metabolismo , Diferenciación Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Microscopía Fluorescente , Mitocondrias/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neurregulina-1/farmacología , Proteínas Proto-Oncogénicas A-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas A-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Serina-Treonina Quinasa 3RESUMEN
Multifunctional nanoparticles that actively target specific cells are promising tools for cancer diagnosis and therapy. In this article we review the synthesis and surface chemistry of Fe-Au nanorods and their characterization using microscopy. The diameter of the rods used in this study was selected to be 150-200 nm so that they did not enter the cells. The 80 nm-long Au tips of the nanorods were functionalized with heregulin (HRG), and the micron-long Fe portion was coated with a poly(ethylene glycol) monolayer to minimize non-specific interactions. Nanorods functionalized with HRG were found to preferentially bind to MCF7 cells that express high levels of the receptor tyrosine-protein kinase ErbB2/3. Magnetic tweezers measurements were used to characterize the kinetic properties of the bond between the HRG on the rods and ErbB2/3 on the surface of the cells. The strong magnetization of Fe-Au nanorods makes them excellent candidates for in-vitro and in-vivo imaging, and magnetic therapeutic applications targeting cancer cells in circulation.
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Nanotubos/química , Neurregulina-1/química , Línea Celular Tumoral , Oro/química , Humanos , Hierro/química , Dispositivos Laboratorio en un Chip , Células MCF-7 , Campos Magnéticos , Pinzas ÓpticasRESUMEN
Advances in genomics and other -omic fields in the last decade have resulted in unprecedented volumes of complex data now being available. These data can enable physicians to provide their patients with care that is more personalized, predictive, preventive and participatory. The expertise required to manage and understand this data is to be found in fields outside of medical science, thus multidisciplinary collaboration coupled to a systems approach is key to unlocking its potential, with concomitant new ways of working. Systems medicine can build on the successes in the field of systems biology, recognizing the human body as the multidimensional network of networks that it is. While systems medicine can provide a conceptual and theoretical framework, its practical goal is to provide physicians the tools necessary for harnessing the rapid advances in basic biomedical science into their routine clinical arsenal.
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Medicina Clínica/tendencias , Genómica/métodos , Biología de Sistemas/métodos , Manejo de la Enfermedad , Humanos , Medicina de Precisión/tendencias , Medicina Preventiva , InvestigaciónAsunto(s)
Antineoplásicos/uso terapéutico , Crisis Blástica/tratamiento farmacológico , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Tirosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Benzamidas , Crisis Blástica/metabolismo , Crisis Blástica/patología , Membrana Celular/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Mesilato de Imatinib , Inmunoprecipitación , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Fosforilación/efectos de los fármacos , Fosfotirosina/análisis , Fosfotirosina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.
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Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Genes ras , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Monocitos/citología , Monocitos/metabolismo , Mutación , Células Mieloides/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/deficiencia , Proteínas de Unión a Fosfatidiletanolamina/genética , PronósticoRESUMEN
Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms-association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS-ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS-ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Proteínas ras/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts. EXPERIMENTAL APPROACH: Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP-/-) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts. KEY RESULTS: RKIP-/- mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP-/- mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP-/- mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1. CONCLUSIONS AND IMPLICATIONS: The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.
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Fibroblastos/enzimología , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Pulmón/enzimología , Proteínas de Unión a Fosfatidiletanolamina/deficiencia , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Animales , Western Blotting , Línea Celular , Proliferación Celular , Enfermedad Crónica , Cricetinae , Cricetulus , Fibroblastos/patología , Eliminación de Gen , Hipertensión Pulmonar/enzimología , Hipoxia/enzimología , Inmunohistoquímica , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Proteínas de Unión a Fosfatidiletanolamina/genética , Fosforilación , Regulación hacia ArribaRESUMEN
The Ras-assocation domain family (RASSF) of tumor suppressor proteins until recently contained six proteins named RASSF1-6. Recently, four novel family members, RASSF7-10, have been identified by homology searches for RA-domain-containing proteins. These additional RASSF members are divergent and structurally distinct from RASSF1-6, containing an N-terminal RA domain and lacking the Sav/RASSF/Hpo (SARAH) domain. Here, we show that RASSF8 is ubiquitously expressed throughout the murine embryo and in normal human adult tissues. Functionally, RNAi-mediated knockdown of RASSF8 in non-small-cell lung cancer (NSCLC) cell lines, increased anchorage-independent growth in soft agar and enhanced tumor growth in severe combined immunodeficiency (SCID) mice. Furthermore, EdU staining of RASSF8-depleted cells showed growth suppression in a manner dependent on contact inhibition. We show that endogenous RASSF8 is not only found in the nucleus, but is also membrane associated at sites of cell-cell adhesion, co-localizing with the adherens junction (AJ) component beta-catenin and binding to E-cadherin. Following RASSF8 depletion in two different lung cancer cell lines using alternative small interfering RNA (siRNA) sequences, we show that AJs are destabilized and E-cadherin is lost from the cell membrane. The AJ components beta-catenin and p65 are also lost from sites of cell-cell contact and are relocalized to the nucleus with a concomitant increase in beta-catenin-dependent and nuclear factor-kappaB (NF-kappaB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actin- cytoskeleton following RASSF8 depletion. Accordingly, scratch wound healing studies show increased cellular migration in RASSF8-deficient cells. These results implicate RASSF8 as a tumor suppressor gene that is essential for maintaining AJs function in epithelial cells and have a role in epithelial cell migration.
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FN-kappa B/fisiología , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteínas Wnt/fisiología , Uniones Adherentes/fisiología , Animales , Cadherinas/fisiología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Citoesqueleto/química , Humanos , Ratones , Ratones SCID , FN-kappa B/genética , Regiones Promotoras Genéticas , Factor de Transcripción ReIA/análisis , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/genética , Xenopus laevisRESUMEN
RASSF2 is a tumour suppressor that in common with the rest of the RASSF family contains Ras association and SARAH domains. We identified the proapoptotic kinases, MST1 and MST2, as the most significant binding partners of RASSF2, confirmed the interactions at endogenous levels and showed that RASSF2 immunoprecipitates active MST1/2. We then showed that RASSF2 can be phosphorylated by a co-immunoprecipitating kinase that is likely to be MST1/2. Furthermore, we showed that RASSF2 and MST2 do indeed colocalize, but whereas RASSF2 alone is nuclear, the presence of MST1 or MST2 results in colocalization in the cytoplasm. Expression of RASSF2 (stably in MCF7 or transiently in HEK-293) increases MST2 levels and knockdown of RASSF2 in HEK-293 cells reduces MST2 levels, in addition colorectal tumour cell lines and primary tumours with low RASSF2 levels show decreased MST2 protein levels. This is likely to be mediated by RASSF2-dependent protection of MST2 against proteolytic degradation. Our findings suggest that MST2 and RASSF2 form an active complex in vivo, in which RASSF2 is maintained in a phosphorylated state and protects MST2 from degradation and turnover. Thus, we propose that the frequent loss of RASSF2 in tumours results in the destablization of MST2 and thus decreased apoptotic potential.
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Apoptosis , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Citoplasma/metabolismo , Epigénesis Genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteómica/métodos , Proteínas Proto-Oncogénicas/metabolismo , Serina-Treonina Quinasa 3 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The authors discuss the role of the Raf kinase inhibitory protein (RKIP) as a modulator of oscillations in NFB signalling. A mathematical model of the NFB signalling pathway was derived and the Lyapunov-Andronov theory was used to analyse dynamical properties of the system. The analytical results were complemented by predictive numerical simulations. Our results suggest that the nature of oscillations, emerging under sustained stimulation of the system, depends on the interplay between the IB kinase (IKK) stimulation and the inhibitory action of RKIP. The authors found a mathematical relation that defines isoclines in IKK and RKIP levels for which the properties of oscillations are conserved and changes in the stimulation can be compensated by modulating RKIP inhibition. On the other hand, the shifting from the current isocline provokes modulation in either the amplitude (for stronger stimulation) or the frequency (for weaker stimulation).
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Quinasa I-kappa B/metabolismo , Modelos Biológicos , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Biología de Sistemas/métodos , Algoritmos , Simulación por Computador , Cinética , Proteínas de Unión a Fosfatidiletanolamina/metabolismoRESUMEN
Lab-on-a-chip systems offer a versatile environment in which low numbers of cells and molecules can be manipulated, captured, detected and analysed. We describe here a microfluidic device that allows the isolation, electroporation and lysis of single cells. A431 human epithelial carcinoma cells, expressing a green fluorescent protein-labelled actin, were trapped by dielectrophoresis within an integrated lab-on-a-chip device containing saw-tooth microelectrodes. Using these same trapping electrodes, on-chip electroporation was performed, resulting in cell lysis. Protein release was monitored by confocal fluorescence microscopy.
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Separación Celular , Electroforesis por Microchip/métodos , Proteómica/métodos , Actinas/análisis , Línea Celular Tumoral , Electroforesis por Microchip/instrumentación , Electroporación , Proteínas Fluorescentes Verdes/análisis , Humanos , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/análisisRESUMEN
AIMS: Raf kinase inhibitory protein (RKIP; also known as PEBP, for phosphatidylethanolamine-binding protein) is an endogenous inhibitor of the Raf- MAPK kinase (MEK)-MAP kinase pathway. It has emerged as a significant metastasis suppressor in a variety of human cancers including colorectal cancer (CRC) and was recently shown to regulate the spindle checkpoint in cultured cells. This study aims at correlating RKIP expression with chromosomal instability in colorectal cancer samples and identifies possible mechanisms of RKIP loss. METHODS: Chromosomal instability was assessed using metaphase-based comparative genomic hybridisation (CGH) and loss of heterozygosity (LOH) in 65 cases with microsatellite stable CRC and correlated with RKIP expression. Methyl-specific PCR was used on DNA extracted from 82 cases with CRC to determine CpG methylation status at the RKIP promoter and the results correlated with RKIP protein expression. RESULTS: We demonstrate for the first time that in microsatellite stable (MSS) CRC, the number of chromosomal losses is inversely proportional to RKIP expression levels. We also show that methylation of the RKIP promoter is a major mechanism by which RKIP expression is silenced in CRC. CONCLUSIONS: RKIP loss by hypermethylation of its promoter could have a significant influence on colorectal cancer aneuploidy, which might explain its association with metastatic progression.
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Neoplasias Colorrectales/metabolismo , Inestabilidad Genómica , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Anciano , Estudios de Cohortes , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Islas de CpG/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas/métodos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Hibridación de Ácido Nucleico/métodos , Proteínas de Unión a Fosfatidiletanolamina/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/metabolismoRESUMEN
Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.
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Proteínas de Unión a Fosfatidiletanolamina/genética , Neoplasias de la Próstata/patología , Factores de Transcripción/fisiología , Transcripción Genética , Bases de Datos Genéticas , Progresión de la Enfermedad , Regulación hacia Abajo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Análisis por Apareamiento , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Proteínas Represoras/fisiología , Factores de Transcripción de la Familia Snail , Transfección , Células Tumorales CultivadasRESUMEN
Cancer can be perceived as a disease of communication between and within cells. The aberrations are pleiotropic, but mitogen-activated protein kinase (MAPK) pathways feature prominently. Here, we discuss recent findings and hypotheses on the role of MAPK pathways in cancer. Cancerous mutations in MAPK pathways are frequently mostly affecting Ras and B-Raf in the extracellular signal-regulated kinase pathway. Stress-activated pathways, such as Jun N-terminal kinase and p38, largely seem to counteract malignant transformation. The balance and integration between these signals may widely vary in different tumours, but are important for the outcome and the sensitivity to drug therapy.
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Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neoplasias/enzimología , Animales , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Neoplasias/genéticaRESUMEN
The Wnt and the extracellular signal regulated-kinase (ERK) pathways are both involved in the pathogenesis of various kinds of cancers. Recently, the existence of crosstalk between Wnt and ERK pathways was reported. Gathering all reported results, we have discovered a positive feedback loop embedded in the crosstalk between the Wnt and ERK pathways. We have developed a plausible model that represents the role of this hidden positive feedback loop in the Wnt/ERK pathway crosstalk based on the integration of experimental reports and employing established basic mathematical models of each pathway. Our analysis shows that the positive feedback loop can generate bistability in both the Wnt and ERK signaling pathways, and this prediction was further validated by experiments. In particular, using the commonly accepted assumption that mutations in signaling proteins contribute to cancerogenesis, we have found two conditions through which mutations could evoke an irreversible response leading to a sustained activation of both pathways. One condition is enhanced production of beta-catenin, the other is a reduction of the velocity of MAP kinase phosphatase(s). This enables that high activities of Wnt and ERK pathways are maintained even without a persistent extracellular signal. Thus, our study adds a novel aspect to the molecular mechanisms of carcinogenesis by showing that mutational changes in individual proteins can cause fundamental functional changes well beyond the pathway they function in by a positive feedback loop embedded in crosstalk. Thus, crosstalk between signaling pathways provides a vehicle through which mutations of individual components can affect properties of the system at a larger scale.
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Transducción de Señal , Proteínas Wnt/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación , Humanos , Sistema de Señalización de MAP Quinasas , Matemática , Modelos Biológicos , Mutación , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismoRESUMEN
Activation of PKA by cAMP agonists, such as 8-Cl-cAMP activation, selectively causes rapid apoptosis in v-abl transformed fibroblasts by inhibiting the Raf-1 kinase. Here we investigated whether 8-Cl-cAMP is useful for the treatment of chronic myelogenous leukaemia (CML), which is hallmarked by the expression of the p210(bcr/abl) oncogene. Autologous bone marrow transplantation is a feasible alternative for patients with no suitable donor, but hampered by the risk of relapse due to the persistence of leukaemia cells in the transplant. To study the effects of 8-Cl-cAMP on primary leukaemic cells, bone marrow cells (BMCs) from eight CML patients (one at diagnosis, three in chronic and four in accelerated phase) were treated. Ex vivo treatment of BMCs obtained in chronic phase of CML with 100 microM 8-Cl-cAMP for 24-48 h led to the selective purging of Philadelphia Chromosome (Ph1 chromosome) without toxic side effects on BMCs from healthy donors as measured by colony-forming unit (CFU) assays. BMCs from patients in accelerated phase showed selective, but incomplete elimination of Ph1 chromosome positive colony forming cells. The mechanism of 8-Cl-cAMP was investigated in FDCP-mix cells transformed by p210(bcr/abl), a cell culture model for CML. The results showed that 8-Cl-cAMP reduced DNA synthesis and viability independent of Raf inhibition as Raf inhibitors had no effect. MEK inhibitors interfered with DNA synthesis, but not with viability. In summary, our results indicate that 8-Cl-cAMP could be useful to purge malignant cells from the bone marrow of patients with CML and certain other forms of leukaemias.
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8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Antineoplásicos/farmacología , Células de la Médula Ósea/fisiología , Purgación de la Médula Ósea/métodos , Trasplante de Médula Ósea , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , ADN/biosíntesis , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Trasplante Autólogo , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
The Raf-MEK-ERK signalling pathway controls fundamental cellular processes including proliferation, differentiation and survival. It remains enigmatic how this pathway can reliably convert a myriad of extracellular stimuli in specific biological responses. Recent results have shown that the Raf family isoforms A-Raf, B-Raf and Raf-1 have different physiological functions. Here we review how Raf isozyme diversity contributes to the specification of functional diversity, in particular regarding the role of Raf isozymes in cancer.