RESUMEN
Non-small cell lung cancers (NSCLCs) establish a highly immunosuppressive tumor microenvironment supporting cancer growth. To interfere with cancer-mediated immunosuppression, selective immune-checkpoint inhibitors (ICIs) have been approved as a standard-of-care treatment for NSCLCs. However, the majority of patients poorly respond to ICI-based immunotherapies. Oncolytic viruses are amongst the many promising immunomodulatory treatments tested as standalone therapy or in combination with ICIs to improve therapeutic outcome. Previously, we demonstrated the oncolytic and immunomodulatory efficacy of low-pathogenic influenza Aviruses (IAVs) against NSCLCs in immunocompetent transgenic mice with alung-specific overexpression of active Raf kinase (Raf-BxB). IAV infection not only resulted in significant primary virus-induced oncolysis, but also caused afunctional reversion of tumor-associated macrophages (TAMs) comprising additional anti-cancer activity. Here we show that NSCLCs as well as TAMs and cytotoxic immune cells overexpress IC molecules of the PD-L2/PD-1 and B7-H3 signaling axes. Thus, we aimed to combine oncolytic IAV-infection with ICIs to exploit the benefits of both anti-cancer approaches. Strikingly, IAV infection combined with the novel B7-H3 ICI led to increased levels of M1-polarized alveolar macrophages and increased lung infiltration by cytotoxic Tlymphocytes, which finally resulted in significantly improved oncolysis of about 80% of existing tumors. In contrast, application of clinically approved α-PD-1 IC antibodies alone or in combination with oncolytic IAV did not provide additional oncolytic or immunomodulatory efficacy. Thus, individualized therapy with synergistically acting oncolytic IAV and B7-H3 ICI might be an innovative future approach to target NSCLCs that are resistant to approved ICIs in patients.
Asunto(s)
Gripe Humana , Neoplasias Pulmonares , Virus Oncolíticos , Orthomyxoviridae , Animales , Humanos , Inhibidores de Puntos de Control Inmunológico , Pulmón , Neoplasias Pulmonares/terapia , Ratones , Virus Oncolíticos/genética , Microambiente TumoralRESUMEN
Although NF-κB is known to play a pivotal role in lung cancer, contributing to tumor growth, microenvironmental changes, and metastasis, the epigenetic regulation of NF-κB in tumor context is largely unknown. Here we report that the IKK2/NF-κB signaling pathway modulates metastasis-associated protein 2 (MTA2), a component of the nucleosome remodeling and deacetylase complex (NuRD). In triple transgenic mice, downregulation of IKK2 (Sftpc-cRaf-IKK2DN) in cRaf-induced tumors in alveolar epithelial type II cells restricted tumor formation, whereas activation of IKK2 (Sftpc-cRaf-IKK2CA) supported tumor growth; both effects were accompanied by altered expression of MTA2. Further studies employing genetic inhibition of MTA2 suggested that in primary tumor growth, independent of IKK2, MTA2/NuRD corepressor complex negatively regulates NF-κB signaling and tumor growth, whereas later dissociation of MTA2/NuRD complex from the promoter of NF-κB target genes and IKK2-dependent positive regulation of MTA2 leads to activation of NF-κB signaling, epithelial-mesenchymal transition, and lung tumor metastasis. These findings reveal a previously unrecognized biphasic role of MTA2 in IKK2/NF-κB-driven primary-to-metastatic lung tumor progression. Addressing the interaction between MTA2 and NF-κB would provide potential targets for intervention of tumor growth and metastasis. SIGNIFICANCE: These findings strongly suggest a prominent role of MTA2 in primary tumor growth, lung metastasis, and NF-κB signaling modulatory functions.
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Histona Desacetilasas/metabolismo , Neoplasias Pulmonares/patología , FN-kappa B/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Animales , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Histona Desacetilasas/genética , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inflamación/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/genética , Proteínas Represoras/genética , Transducción de Señal , Transactivadores/genética , Microambiente TumoralRESUMEN
Non-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer and demonstrates high resistance to radiation and chemotherapy. These tumors evade immune system detection by promoting an immunosuppressive tumor microenvironment. Genetic analysis has revealed oncogenic activation of the Ras/Raf/MEK/ERK signaling pathway to be a hallmark of NSCLCs, which promotes influenza A virus (IAV) infection and replication in these cells. Thus, we aimed to unravel the oncolytic properties of IAV infection against NSCLCs in an immunocompetent model in vivo. Using Raf-BxB transgenic mice that spontaneously develop NSCLCs, we demonstrated that infection with low-pathogenic IAV leads to rapid and efficient oncolysis, eliminating 70% of the initial tumor mass. Interestingly, IAV infection of Raf-BxB mice caused a functional reversion of immunosuppressed tumor-associated lung macrophages into a M1-like pro-inflammatory active phenotype that additionally supported virus-induced oncolysis of cancer cells. Altogether, our data demonstrate for the first time in an immunocompetent in vivo model that oncolytic IAV infection is capable of restoring and redirecting immune cell functions within the tumor microenvironment of NSCLCs.
RESUMEN
RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons.
Asunto(s)
Diferenciación Celular , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Animales Recién Nacidos , Proliferación Celular/genética , Giro Dentado/citología , Giro Dentado/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Neurogénesis/genética , Neuronas/citología , Proteínas Proto-Oncogénicas c-raf/genética , Factores de TiempoRESUMEN
Identifying metastatic tumor growth at an early stage has been one of the biggest challenges in the treatment of lung cancer. By genetic lineage tracing approach in a conditional model of Non-Small Cell Lung Cancer (NSCLC) in mice, we demonstrate that cystic lesions represent an early stage of metastatic invasion. We generated a mouse model for NSCLC which incorporated a heritable DsRed fluorescent tag driven by the ubiquitous CAG promoter in the alveolar type II cells of the lung. We found early cystic lesions in a secondary organ (liver) that lacked the expression of bona fide lung makers namely Scgb1a1 and surfactant protein C Sftpc and were DsRed positive hence identifying lung as their source of origin. This demonstrates the significant potential of alveolar type II cells in orchestrating the process of metastasis, rendering it as one of the target cell types of the lung of therapeutic importance in human NSCLC.
RESUMEN
Dyspnea is a frequent, devastating, and poorly understood symptom of advanced lung cancer. In our cohort, among 519 patients who underwent a computed tomography scan for the diagnosis of lung cancer, 250 had a mean pulmonary artery diameter of >28 mm, indicating pulmonary hypertension (PH). In human lung cancer tissue, we consistently observed increased vascular remodeling and perivascular inflammatory cell accumulation (macrophages/lymphocytes). Vascular remodeling, PH, and perivascular inflammatory cell accumulation were mimicked in three mouse models of lung cancer (LLC1, KRasLA2 , and cRaf-BxB). In contrast, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ immunodeficient xenograft and dominant-negative IKK2 mutant triple transgenic (Sftpc-rtTA/Tet-O-Ikk2DN) mice did not develop PH. Coculturing human lung cancer cells with macrophages and lymphocytes strongly up-regulated cytokine release, provoking enhanced migration, apoptosis resistance, and phosphodiesterase 5 (PDE5)-mediated up-regulation of human lung vascular cells, which are typical features of PH. The PDE5 inhibitor sildenafil largely suppressed PH in the LLC1 model. We conclude that lung cancer-associated PH represents a distinct PH category; targeting inflammation in the microenvironment and PDE5 offers a potential therapeutic option.
Asunto(s)
Disnea/fisiopatología , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/fisiopatología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/fisiopatología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Células Dendríticas , Disnea/inmunología , Ecocardiografía , Humanos , Hipertensión Pulmonar/etiología , Inmunohistoquímica , Técnicas In Vitro , Inflamación/inmunología , Inflamación/fisiopatología , Neoplasias Pulmonares/complicaciones , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Linfocitos T/metabolismoRESUMEN
Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Early LC diagnosis is crucial to reduce the high case fatality rate of this disease. In this case-control study, we developed an accurate LC diagnosis test using retrospectively collected formalin-fixed paraffin-embedded (FFPE) human lung tissues and prospectively collected exhaled breath condensates (EBCs). Following international guidelines for diagnostic methods with clinical application, reproducible standard operating procedures (SOP) were established for every step comprising our LC diagnosis method. We analyzed the expression of distinct mRNAs expressed from GATA6 and NKX2-1, key regulators of lung development. The Em/Ad expression ratios of GATA6 and NKX2-1 detected in EBCs were combined using linear kernel support vector machines (SVM) into the LC score, which can be used for LC detection. LC score-based diagnosis achieved a high performance in an independent validation cohort. We propose our method as a non-invasive, accurate, and low-price option to complement the success of computed tomography imaging (CT) and chest X-ray (CXR) for LC diagnosis.
Asunto(s)
Pruebas Respiratorias/métodos , Factor de Transcripción GATA6/análisis , Neoplasias Pulmonares/diagnóstico , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Estudios de Casos y Controles , Técnicas de Apoyo para la Decisión , Humanos , Neoplasias Pulmonares/patología , Estudios Prospectivos , Isoformas de Proteínas/análisis , Estudios Retrospectivos , Factor Nuclear Tiroideo 1RESUMEN
Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.
Asunto(s)
Membrana Celular/metabolismo , Mutación de Línea Germinal , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Factor de Transcripción STAT3/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exones/genética , Femenino , Técnicas de Sustitución del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/química , Transducción de SeñalRESUMEN
The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K(+) channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf (+/-) mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.
Asunto(s)
Oído Interno/metabolismo , Pérdida Auditiva/genética , Ruido , Proteínas Proto-Oncogénicas c-raf/fisiología , Animales , Apoptosis/genética , Cóclea/metabolismo , Oído Interno/embriología , Femenino , Pérdida Auditiva/metabolismo , Masculino , Ratones , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de SeñalRESUMEN
Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.
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Antineoplásicos/farmacología , Triterpenos/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Triterpenos/administración & dosificación , Triterpenos/síntesis química , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.
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Adenoma/enzimología , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/enzimología , Neoplasias Pulmonares/enzimología , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Alveolos Pulmonares/enzimología , Mucosa Respiratoria/enzimología , Adenoma/genética , Adenoma/patología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Epiteliales/patología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Alveolos Pulmonares/patología , Mucosa Respiratoria/patologíaRESUMEN
In metazoans, the highly conserved MAPK signaling pathway regulates cell fate decision. Aberrant activation of this pathway has been implicated in multiple human cancers and some developmental disorders. KSR1 functions as an essential scaffold that binds the individual components of the cascade and coordinates their assembly into multiprotein signaling platforms. The mechanism of KSR1 regulation is highly complex and not completely understood. In this study, we identified Tyr(728) as a novel regulatory phosphorylation site in KSR1. We show that Tyr(728) is phosphorylated by LCK, uncovering an additional and unexpected link between Src kinases and MAPK signaling. To understand how phosphorylation of Tyr(728) may regulate the role of KSR1 in signal transduction, we integrated structural modeling and biochemical studies. We demonstrate that Tyr(728) is involved in maintaining the conformation of the KSR1 kinase domain required for binding to MEK. It also affects phosphorylation and activation of MEK by RAF kinases and consequently influences cell proliferation. Moreover, our studies suggest that phosphorylation of Tyr(728) may affect the intrinsic kinase activity of KSR1. Together, we propose that phosphorylation of Tyr(728) may regulate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Activación Enzimática , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Tirosina/químicaRESUMEN
This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.
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Diferenciación Celular/genética , Cerebelo/metabolismo , Hipocampo/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Animales Recién Nacidos , Conducta Animal , Cerebelo/crecimiento & desarrollo , Giro Dentado/crecimiento & desarrollo , Giro Dentado/metabolismo , Eliminación de Gen , Hipocampo/crecimiento & desarrollo , Masculino , Ratones , Ratones Transgénicos , FenotipoRESUMEN
Human lung cancer is a disease with high incidence and accounts for most cancer-related deaths in both men and women. Metastasis is a common event in non-small cell lung carcinoma (NSCLC), diminishing the survival chance of the patients with this type of tumor. It has been shown that MYC is involved in the development of metastasis from NSCLC, but the mechanism underlying this switch remained to be identified. Here, we focus on GATA4 as a MYC target in the development of metastasis with origin in lung adenocarcinoma, the most common type of NSCLC. Epigenetic alterations at the GATA4 promoter level were observed after MYC expression in lung adenocarcinoma in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. Our results thus identify a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in lung adenocarcinoma, suggesting novel potential targets for the development of antimetastatic therapy.
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Adenocarcinoma/genética , Factor de Transcripción GATA4/genética , Genes myc , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-myc/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Adhesión Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Metilación de ADN , Proteínas de Unión al ADN/genética , Epigénesis Genética , Femenino , Factor de Transcripción GATA4/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Mucina 2/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas p21(ras) , Factores de Transcripción/genética , Proteínas ras/genéticaRESUMEN
BACKGROUND: Gene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1). The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs), all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3, a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription. RESULTS: Our current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the existence of MK-mediated negative feedback regulation on pERK. CONCLUSION: We here identify and characterize important actors in a PRC1-dependent epigenetic signal/response mechanism, some of which appear to be nonspecific global responses, whereas others provide modular specificity. Our findings provide novel insight into a Polycomb-mediated epigenetic mechanism that dynamically controls gene transcription and support a direct link between PRC1 and cellular responses to changes in the microenvironment.
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Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Proteínas Inhibidoras de la Apoptosis/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Estabilidad de Enzimas/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Melanoma/genética , Melanoma/patología , Proteínas de Neoplasias/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-raf/genéticaRESUMEN
The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoesqueleto/metabolismo , Dimerización , Genes Supresores de Tumor/fisiología , Humanos , Complejos Multiproteicos/metabolismo , Prenilación/fisiología , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2(high) and HER2(low) cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2(low) cell fraction. Accordingly, HER2(low) cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2(low) sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2(high) fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cartilla de ADN , Femenino , Humanos , Piranos/administración & dosificación , Piranos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , TrastuzumabRESUMEN
Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body.associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways.