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1.
Nature ; 546(7660): 676-680, 2017 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-28658220

RESUMEN

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole-body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a 'MetAlert' discovery platform for drivers and inhibitors of metastasis.


Asunto(s)
Citocinas/metabolismo , Vasos Linfáticos/metabolismo , Metástasis de la Neoplasia/diagnóstico por imagen , Metástasis de la Neoplasia/patología , Imagen de Cuerpo Entero/métodos , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Femenino , Genes Reporteros , Humanos , Linfangiogénesis , Vasos Linfáticos/patología , Masculino , Melanoma/diagnóstico por imagen , Melanoma/metabolismo , Melanoma/patología , Ratones , Midkina , Comunicación Paracrina , Pronóstico , Recurrencia , Reproducibilidad de los Resultados , Serina-Treonina Quinasas TOR/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/análisis , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
2.
EMBO Mol Med ; 13(12): e12924, 2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34762341

RESUMEN

Long-range communication between tumor cells and the lymphatic vasculature defines competency for metastasis in different cancer types, particularly in melanoma. Nevertheless, the discovery of selective blockers of lymphovascular niches has been compromised by the paucity of experimental systems for whole-body analyses of tumor progression. Here, we exploit immunocompetent and immunodeficient mouse models for live imaging of Vegfr3-driven neolymphangiogenesis, as a versatile platform for drug screening in vivo. Spatiotemporal analyses of autochthonous melanomas and patient-derived xenografts identified double-stranded RNA mimics (dsRNA nanoplexes) as potent inhibitors of neolymphangiogenesis, metastasis, and post-surgical disease relapse. Mechanistically, dsRNA nanoplexes were found to exert a rapid dual action in tumor cells and in their associated lymphatic vasculature, involving the transcriptional repression of the lymphatic drivers Midkine and Vegfr3, respectively. This suppressive function was mediated by a cell-autonomous type I interferon signaling and was not shared by FDA-approved antimelanoma treatments. These results reveal an alternative strategy for targeting the tumor cell-lymphatic crosstalk and underscore the power of Vegfr3-lymphoreporters for pharmacological testing in otherwise aggressive cancers.


Asunto(s)
Melanoma , ARN Bicatenario , Animales , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Ratones Desnudos , Transducción de Señal
3.
Nat Med ; 26(12): 1865-1877, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33077955

RESUMEN

An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.


Asunto(s)
Carcinogénesis/efectos de los fármacos , Melanoma Experimental/terapia , Midkina/genética , Microambiente Tumoral/genética , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Terapia Genética , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Midkina/farmacología , FN-kappa B/genética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Transcriptoma/genética
4.
Cancer Cell ; 35(1): 46-63.e10, 2019 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-30581152

RESUMEN

Modulators of mRNA stability are not well understood in melanoma, an aggressive tumor with complex changes in the transcriptome. Here we report the ability of p62/SQSTM1 to extend mRNA half-life of a spectrum of pro-metastatic factors. These include FERMT2 and other transcripts with no previous links to melanoma. Transcriptomic, proteomic, and interactomic analyses, combined with validation in clinical biopsies and mouse models, identified a selected set of RNA-binding proteins (RBPs) recruited by p62, with IGF2BP1 as a key partner. This p62-RBP interaction distinguishes melanoma from other tumors where p62 controls autophagy or oxidative stress. The relevance of these data is emphasized by follow-up analyses of patient prognosis revealing p62 and FERMT2 as adverse determinants of disease-free survival.


Asunto(s)
Melanoma/metabolismo , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , ARN Mensajero/química , Proteínas de Unión al ARN/metabolismo , Proteína Sequestosoma-1/metabolismo , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Proteínas de la Membrana/química , Ratones , Proteínas de Neoplasias/química , Trasplante de Neoplasias , Mapas de Interacción de Proteínas , Proteómica/métodos , Estabilidad del ARN , Análisis de Matrices Tisulares
5.
Mol Biol Cell ; 15(12): 5583-92, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15371543

RESUMEN

Retinoic acid (RA) is a potent regulator of neuronal cell differentiation. RA normally activates gene expression by binding to nuclear receptors that interact with response elements (RAREs) in regulatory regions of target genes. We show here that in PC12 cell subclones in which the retinoid causes neurite extension, RA induces a rapid and sustained phosphorylation of CREB (cyclic AMP response element binding protein), compatible with a nongenomic effect. RA also causes a rapid increase of CREB phosphorylation in primary cultures of cerebrocortical cells and of dorsal root ganglia neurons from rat embryos. RA-mediated phosphorylation of CREB leads to a direct stimulation of CREB-dependent transcriptional activity and to activation of the expression of genes such as c-fos, which do not contain RAREs but contain cAMP response elements (CREs) in their promoters. CREB is a major target of extracellular signal regulated kinase ERK1/2 signaling in neuronal cells, and we demonstrate here that RA induces an early stimulation of ERK1/2, which is required both for CREB phosphorylation and transcriptional activity. These results demonstrate that RA, by a nongenomic mechanism, stimulates signaling pathways that lead to phosphorylation of transcription factors, which in turn activate the transcription of genes involved in neuronal differentiation.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/citología , Células PC12 , Fosforilación/efectos de los fármacos , Ratas , Elementos de Respuesta/genética , Factores de Tiempo , Transcripción Genética/efectos de los fármacos
6.
Nat Commun ; 8(1): 2249, 2017 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-29269732

RESUMEN

Melanomas are well-known for their altered mRNA expression profiles. Yet, the specific contribution of mRNA binding proteins (mRBPs) to melanoma development remains unclear. Here we identify a cluster of melanoma-enriched genes under the control of CUGBP Elav-like family member 1 (CELF1). CELF1 was discovered with a distinct prognostic value in melanoma after mining the genomic landscape of the 692 known mRBPs across different cancer types. Genome-wide transcriptomic, proteomic, and RNA-immunoprecipitation studies, together with loss-of-function analyses in cell lines, and histopathological evaluation in clinical biopsies, revealed an intricate repertoire of CELF1-RNA interactors with minimal overlap with other malignancies. This systems approach uncovered the oncogene DEK as an unexpected target and downstream effector of CELF1. Importantly, CELF1 and DEK were found to represent early-induced melanoma genes and adverse indicators of overall patient survival. These results underscore novel roles of CELF1 in melanoma, illustrating tumor type-restricted functions of RBPs in cancer.


Asunto(s)
Proteínas CELF1/fisiología , Melanoma/genética , Oncogenes , Biología de Sistemas , Regiones no Traducidas 3' , Biopsia , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas Cromosómicas no Histona/metabolismo , Humanos , Inmunoprecipitación , Melanoma/patología , Proteínas Oncogénicas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Pronóstico , Proteómica , ARN Neoplásico/genética , Análisis de Supervivencia , Transcriptoma
7.
Autophagy ; 12(10): 1776-1790, 2016 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-27464255

RESUMEN

Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.


Asunto(s)
Proteína 5 Relacionada con la Autofagia/genética , Pérdida de Heterocigocidad/genética , Melanoma/genética , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Mapeo Cromosómico , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heterocigoto , Ratones , Metástasis de la Neoplasia , Nevo/genética , Nevo/patología , Pigmentación/genética , Pronóstico , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas B-raf/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Riesgo , Análisis de Supervivencia
8.
Nat Commun ; 7: 13418, 2016 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-27857118

RESUMEN

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.


Asunto(s)
Melanoma/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Melanoma/genética , Ratones , Neoplasias Experimentales , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética
9.
Oncotarget ; 6(14): 11848-62, 2015 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-26008978

RESUMEN

Derailed endolysosomal trafficking is emerging as a widespread feature of aggressive neoplasms. However, the oncogenic signals that alter membrane homeostasis and their specific contribution to cancer progression remain unclear. Understanding the upstream drivers and downstream regulators of aberrant vesicular trafficking is distinctly important in melanoma. This disease is notorious for its inter- and intra-tumoral heterogeneity. Nevertheless, melanomas uniformly overexpress a cluster of endolysosomal genes, being particularly addicted to the membrane traffic regulator RAB7. Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined. Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells. This strategy revealed Class I PI3K as the key trigger of a hyperactive influx of macropinosomes that melanoma cells counteract via RAB7-mediated lysosomal degradation. In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes. Together, these results provide new insight into the regulators and modes of action of RAB7, broadening the impact of endosomal fitness on melanoma development.


Asunto(s)
Melanoma/metabolismo , Melanoma/patología , Pinocitosis/fisiología , Proteínas de Unión al GTP rab/metabolismo , Animales , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Xenoinjertos , Humanos , Immunoblotting , Ratones , Microscopía Electrónica de Transmisión , Fosfatidilinositol 3-Quinasas/metabolismo , Transfección , Proteínas de Unión a GTP rab7
10.
Cancer Cell ; 26(1): 61-76, 2014 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-24981740

RESUMEN

Although common cancer hallmarks are well established, lineage-restricted oncogenes remain less understood. Here, we report an inherent dependency of melanoma cells on the small GTPase RAB7, identified within a lysosomal gene cluster that distinguishes this malignancy from over 35 tumor types. Analyses in human cells, clinical specimens, and mouse models demonstrated that RAB7 is an early-induced melanoma driver whose levels can be tuned to favor tumor invasion, ultimately defining metastatic risk. Importantly, RAB7 levels and function were independent of MITF, the best-characterized melanocyte lineage-specific transcription factor. Instead, we describe the neuroectodermal master modulator SOX10 and the oncogene MYC as RAB7 regulators. These results reveal a unique wiring of the lysosomal pathway that melanomas exploit to foster tumor progression.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Linaje de la Célula , Lisosomas/enzimología , Melanoma/enzimología , Neoplasias Cutáneas/enzimología , Proteínas de Unión al GTP rab/metabolismo , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/mortalidad , Melanoma/secundario , Melanoma/terapia , Ratones , Invasividad Neoplásica , Estadificación de Neoplasias , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Factores de Tiempo , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7
11.
Cancer Cell ; 16(2): 103-14, 2009 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-19647221

RESUMEN

Inappropriate drug delivery, secondary toxicities, and persistent chemo- and immunoresistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic double-stranded RNA (dsRNA) and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid, coadministered with polyethyleneimine as carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes, and apoptotic factors.


Asunto(s)
Apoptosis/inmunología , Autofagia/inmunología , Inmunidad Innata , Melanoma/inmunología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Línea Celular Tumoral , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Endosomas/efectos de los fármacos , Endosomas/genética , Endosomas/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1 , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/metabolismo , Melanoma/patología , Melanoma/terapia , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Fagosomas/efectos de los fármacos , Fagosomas/genética , Fagosomas/metabolismo , Poli C/farmacología , Polietileneimina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Bicatenario
12.
J Biol Chem ; 279(31): 32813-23, 2004 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-15173174

RESUMEN

ERKs, mitogen-activated protein kinases, are well characterized as key mediators in the conveyance of signals that promote cell survival in cells of hemopoietic origin, a key factor in the upbringing of leukemogenesis. It is also well known that ERKs phosphorylate a wide array of substrates distributed throughout distinct cellular locations such as the nucleus, cytoplasm, and cell periphery, but the relative contribution of these compartmentalized signal components to the overall survival signal generated by activation of ERKs has yet to be established. To this end, we have utilized constitutively activated forms of ERK2, whose expression is restricted to the nucleus or to the cytoplasm, to investigate the consequences of compartmentalized activation of ERK in the survival of chronic myelogenous leukemia cells subjected to distinct apoptogenic stimuli. We show that cytoplasmic ERK2 activity protected against apoptosis caused by prolonged serum starvation, whereas ERK2 activation restricted to the nucleus antagonized apoptosis induced by the Bcr-Abl inhibitor STI571. On the other hand, neither cytoplasmic nor nuclear ERK2 activities were effective in counteracting apoptosis induced by UV light. These results demonstrate that the protective effects of ERK2 against defined apoptogenic stimuli are strictly dependent on the cellular localization where ERK activation takes place. Furthermore, we present evidence suggesting that the complex I kappa B-NF kappa B participates on ERK2-mediated survival mechanisms, in a fashion dependent on the cellular location where ERK2 is active and on the causative apoptogenic stimulus.


Asunto(s)
Apoptosis , Leucemia/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Medio de Cultivo Libre de Suero/farmacología , Citoplasma/metabolismo , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Immunoblotting , Células K562 , Leucemia Mieloide/metabolismo , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación , Plásmidos/metabolismo , Fracciones Subcelulares , Factores de Tiempo , Transfección , Rayos Ultravioleta
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