Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics.

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.The authors thank previous and present colleagues in the CNIO Melanoma Group, particularly Damia Tormo and Lisa Osterloh for help and support at the initial stages of this study; Jose A Esteban (CSIC-UAM) for critical reading of this manuscript; Lionel Larue (INSERM; France) and Martin McMahon (Hunstman Cancer Center, USA) for the Tyr:CreERT2 and BrafCA mouse strains, respectively; and Ignacio Melero at Hospital Clinico, Pamplona, Spain, for Ifnar1-deficient mice. The authors thank Isabel Blanco, Soraya Ruiz, and Virginia Granda (CNIO-Animal Facility Unit), Diego Megias (CNIO-Confocal Unit), and Eduardo Jose Caleiras and Patricia Gonzalez (CNIO-Histopathology Unit) for technical assistance. M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2017-89533-R), the Asociacion Espanola Contra el Cancer (AECC), Fundacion La Caixa, and an Established Investigator Award by the Melanoma Research Alliance (MRA). D.O. is funded by grants from the Spanish Ministry of Health (AES-PIS PI18/1057) and "Beca Leonardo a Investigadores y Creadores Culturales 2018 de la Fundacion BBVA". The CNIO Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019. S.O. is also supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-71376-R).S

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5

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.M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (projects SAF2011-28317, SAF2014-56868-R and RTC-2014-2442-1), as well as a Team Science Award by the Melanoma Research Alliance, and grants from the Worldwide Cancer Research and the Asociacion Espanola Contra el Cancer (AECC). M.G-F was funded by a Juan de la ~ Cierva postdoctoral fellowship from the Spanish Ministry of Education and P.K and M.C. by predoctoral fellowships from Fundación La Caixa

p62/SQSTM1 Fuels Melanoma Progression by Opposing mRNA Decay of a Selective Set of Pro-metastatic Factors

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.M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2014-56868-R; SAF2017-89533-R), the Asociación Española Contra el Cáncer (AECC), TV’13-20131430 (Marato de TV3), the Worldwide Cancer Research, an Established Investigator Award by the Melanoma Research Alliance (MRA), and a L'Oreal-Paris USA-MRA Team Science Award for Women in Scientific Research. M.S.S. also acknowledges a donation from “Fundación Causa Alexandra”, Spain. P.K. was a recipient of a predoctoral fellowship from Fundación La Caixa. E.R.-F. was funded by Fundación Mutua Madrileña (FMM-2013) and was a recipient of a fellowship from ‘‘Fundación Científica de la Asociación Española Contra el Cáncer”. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P. is funded by FIS 2014/173711/02568 and CIBERONC, and P.L.O.-R. by FIS 11/17592014/01784, from the Spanish Ministry of Health

RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endolysosomal Pathway

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.M.S.S. is funded by Projects SAF2011-28317 and Consolider RNAREG from the Spanish Ministry of Economy and Innovation, R01CA125017 from the NIH, and a Team Science Award by the Melanoma Research Foundation. J.L.R.-P. and P.O.-R. are funded by grants FIS 11/025685 and FIS 11/1759, respectively, from the Spanish Ministry of Health. J.L.R.-P. was also supported by grant FMM-2008-106 of Fundación Mutua Madrileña, and P.O.-R. by Red Tematica de Investigacion Cooperativa en Cancer. D.A.-C. and E.P.-G. are recipients of Scientists in Training predoctoral fellowships from the Spanish Ministry of Science and Innovation. M.C. and P.K. are funded by predoctoral fellowships of Fundación La Caixa. E.R.-F. is the recipient of a postdoctoral fellowship from Fundación Científica de la Asociación Española Contra el Cáncer, and J.A.J. and H.-W.W. are funded by the American Cancer Society (RSG-12-076-01-LIB)

Activación de CREB y regulación de genes tempranos por ácido retinoico en el subclón A126-1B2

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular.El ácido retinoico todo trans m),ju ega un importante papel en la proliferación y diferenciación de diversos tipos celulares. Las acciones del RA están mediadas por la interacción con sus receptores nucleares, que regulan la transcripción de un gran número de genes uniéndose a los elementos de respuesta @ARES) localizados en sus regiones promotoras. En esta tesis demostramos que el RA puede ejercer también acciones extragenómicas que conducen a una rápida estimulación de la actividad de ciertas quinasas claves para la fosforilación de factores de transcripcíón, que a su vez intervienen en la inducción de la actividad de los promotores de díversos genes. El RA causa diferenciación neuronal con extensión de neurítas en las células A126-1B2 derivadas de las PC12, que se caracterizan por expresar un nivel elevado de receptores de retinoides. Estos cambios morfológicos son similares a los producídos por el NGF, el agente diferenciador por excelencia en las células PC12. Hemos demostrado que en células A126-1B2 el RA induce una rápida activación de los promotores de los genes c-fos, c-Jun y jun-B que carecen de RAREs, y hemos comprobado que la activación del gen cifos por RA requiere la participación del factor de transcripción CREB. El RA causa una rápida fosforilación de CREB, compatible con un mecanismo de regulación de tipo extragenómico. Este factor, esencial en la regulación transcripcional que acompaña al proceso de diferenciación neuronal, es fosforilado en respuesta al RA con una intensidad similar a la observada en respuesta al NGF y ambos agentes parecen actuar a través de las mismas rutas de señalización. Este efecto del RA es reproducido por un ligando específico del receptor de RA, RAR, sugiriendo la participación de este receptor en dicho proceso. La fosforilación inducida por RA se traduce en la activación de la capacidad transactivadora de CREB. Por otro lado, hemos visto que el RA induce también rápida e intensamente la activación de las ERK112. Dicha activación parece ser clave tanto para la fosforilación de CREB como para la activación del promotor de c-fos mediada por el RA. La PKC y el calcio intracelular parecen ser importantes también en la regulación de ambos procesos. Estos datos aportan la primera evidencia de actuación extragenómica del RA a través de la interacción con rutas de señalización normalmente activadas en respuesta a agentes como el NGF que inducen la diferenciación de las células PC12. Los elevados niveles de receptores de RA, así como la deficiencia en PKA de las células A126-1B2, parecen ser factores significativos en la respuesta diferenciadora al RA.Peer reviewe

Activación de CREB y regulación de genes tempranos por ácido retinoico en el subclón A126-1B2 de células PC12

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-10-2001El ácido retinoico todo trans m),ju ega un importante papel en la proliferación y diferenciación de diversos tipos celulares. Las acciones del RA están mediadas por la interacción con sus receptores nucleares, que regulan la transcripción de un gran número de genes uniéndose a los elementos de respuesta @ARES) localizados en sus regiones promotoras. En esta tesis demostramos que el RA puede ejercer también acciones extragenómicas que conducen a una rápida estimulación de la actividad de ciertas quinasas claves para la fosforilación de factores de transcripcíón, que a su vez intervienen en la inducción de la actividad de los promotores de díversos genes. El RA causa diferenciación neuronal con extensión de neurítas en las células A126-1B2 derivadas de las PC12, que se caracterizan por expresar un nivel elevado de receptores de retinoides. Estos cambios morfológicos son similares a los producídos por el NGF, el agente diferenciador por excelencia en las células PC12. Hemos demostrado que en células A126-1B2 el RA induce una rápida activación de los promotores de los genes c-fos, c-Jun y jun-B que carecen de RAREs, y hemos comprobado que la activación del gen cifos por RA requiere la participación del factor de transcripción CREB. El RA causa una rápida fosforilación de CREB, compatible con un mecanismo de regulación de tipo extragenómico. Este factor, esencial en la regulación transcripcional que acompaña al proceso de diferenciación neuronal, es fosforilado en respuesta al RA con una intensidad similar a la observada en respuesta al NGF y ambos agentes parecen actuar a través de las mismas rutas de señalización. Este efecto del RA es reproducido por un ligando específico del receptor de RA, RAR, sugiriendo la participación de este receptor en dicho proceso. La fosforilación inducida por RA se traduce en la activación de la capacidad transactivadora de CREB. Por otro lado, hemos visto que el RA induce también rápida e intensamente la activación de las ERK112. Dicha activación parece ser clave tanto para la fosforilación de CREB como para la activación del promotor de c-fos mediada por el RA. La PKC y el calcio intracelular parecen ser importantes también en la regulación de ambos procesos. Estos datos aportan la primera evidencia de actuación extragenómica del RA a través de la interacción con rutas de señalización normalmente activadas en respuesta a agentes como el NGF que inducen la diferenciación de las células PC12. Los elevados niveles de receptores de RA, así como la deficiencia en PKA de las células A126-1B2, parecen ser factores significativos en la respuesta diferenciadora al RA

Rapid Effects of Retinoic Acid on CREB and ERK Phosphorylation in Neuronal Cells

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

Subcellular localization determines the protective effects of activated ERK2 against distinct apoptogenic stimuli in myeloid leukemia cells

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 IκB-NFκB participates on ERK2-mediated survival mechanisms, in a fashion dependent on the cellular location where ERK2 is active and on the causative apoptogenic stimulus.This work was supported by a grant from Fundación Marcelino Botín, Spanish Ministry of Education Grants BMC2002-0102 (to P. C.) and SAF02-4193 (to J. L.), and by Spanish Ministry of Health Grant FIS PI020774 (to R. P.).Peer Reviewe