AKT activation seems to be associated with apoptotic signals and not with pro-survival signals in a pristane-induced lupus model.

Several studies have shown that in addition to its role as a survival factor and tumor promoting agent, AKT is also able to exhibit pro-apoptotic effects under diverse conditions, including oxidative stress, cytokine stimulation and exposure to cytotoxic chemicals like staurosporine, methotrexate, docetaxel and etoposide. Moreover, phosphorylation of second mitochondria-derived activator of caspases (SMAC) by AKT promotes caspase-3 activation during etoposide-induced apoptosis in HeLa cells. Our data show that injection of pristane into the peritoneum induces apoptosis-mediated cell death of peritoneal exudate cells (PECs), as evidenced by the increased number of annexin V+ peritoneal cells and their increased levels of cleaved/active caspase-3. Indeed, the higher levels of activated caspase-3 protein in WT PECs, particularly at 2-weeks post pristane treatment, are indicative of a higher rate of apoptosis compared to Cd38¿/¿ cells. In contrast, no differences were observed in the levels of MCL-1, an anti-apoptotic protein and member of the BCL2 family. Furthermore, kinases ERK1/2 and AKT showed distinct activation kinetics in pristane-elicited PECs. Interestingly, caspase-3 activation followed similar kinetics to AKT activation in both WT and Cd38¿/¿ PECs, while ERK activation correlated with increased levels of MCL-1. In summary our data strongly suggest that in the pristane-induced lupus model AKT activation is associated with apoptotic signals and not with survival signals. Further studies, however, are required to identify specific pro- and anti-apoptotic target proteins that are phosphorylated by ERK or AKT following pristane treatment, and that regulate the apoptotic process

Mice deficient in CD38 develop an attenuated form of collagen type II-induced arthritis

CD38, a type II transmembrane glycoprotein expressed in many cells of the immune system, is involved in cell signaling, migration and differentiation. Studies in CD38 deficient mice (CD38 KO mice) indicate that this molecule controls inflammatory immune responses, although its involvement in these responses depends on the disease model analyzed. Here, we explored the role of CD38 in the control of autoimmune responses using chicken collagen type II (col II) immunized C57BL/6-CD38 KO mice as a model of collagen-induced arthritis (CIA). We demonstrate that CD38 KO mice develop an attenuated CIA that is accompanied by a limited joint induction of IL-1β and IL-6 expression, by the lack of induction of IFNγ expression in the joints and by a reduction in the percentages of invariant NKT (iNKT) cells in the spleen. Immunized CD38 KO mice produce high levels of circulating IgG1 and low of IgG2a anti-col II antibodies in association with reduced percentages of Th1 cells in the draining lymph nodes. Altogether, our results show that CD38 participates in the pathogenesis of CIA controlling the number of iNKT cells and promoting Th1 inflammatory responses

Dissecting and deactivating inmunosuppressive signals in melanoma microenvironment

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 22-02-2019Esta tesis tiene embargado el acceso al texto completo hasta el 22-08-2020Cutaneous melanoma is the most lethal form of skin cancer, characterized by a high metastatic potential and a remarkable ability to evade immune surveillance. Immune-based therapies using Immune Checkpoint blockers (ICB; i.e anti-PD1/PD-L1, anti-CTLA4) are currently the most successful treatments for metastatic melanoma. Unfortunately, clinical responses to ICB remain limited and/or transient. Resistance to ICBs is often observed in patients with low immunogenic tumors (cold tumors), or lesions with high infiltration of immune suppressive cells, such as tumor-associated macrophages (TAMs), T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Mechanisms that favor the recruitment and modulation of these immunosuppressive cells, and importantly, how to deactivate them are pending questions with direct translational impact in the melanoma field. This Thesis project was set to (i) characterize mechanisms of resistance to ICB in melanoma, with particular emphasis on myeloid suppressive cells (TAMs and MDSC), and (ii) to screen for novel therapeutic agents able to rewire myeloid cells towards potent anti-tumor responses. To this end, proteomic and transcriptomic analyses were performed to identify novel melanoma-secreted factors involved in tumor metastasis and drug resistance. In particular, we focused on the heparin-binding factor MIDKINE (MDK), a tumor-secreted factor that promotes lymphangiogenesis and metastasis. Using a combination of in vivo and in vitro experiments, we can now conclude that MDK activates NF-κB in melanoma cells in an ALK-dependent manner, leading to an inflammatory secretory program that increases intratumoral recruitment of MDSCs and Tregs. Additionally, MDK-induced secretome was found to favor STAT3 phosphorylation in myeloid cells, promoting their immune suppressive polarization. By modulating MDK levels of expression, we have shown that MDK secretion prevents immunotherapy responses to ICB in vivo. Moreover, MDK expression correlated with poor response to immunotherapy in melanoma patients, evidencing the relevance of our results. In parallel, we have observed that dsRNA-based nanoparticles (BO-110) can reverse this immunosuppression by a combined action in melanoma cells and macrophages. Specifically, we demonstrated that BO-110 suppresses MDK expression in melanoma cells, in a Type I IFN dependent manner, and induces their immunogenic cell death. In macrophages, BO-110 induced a potent tumoricidal activation leading to tumor-specific immune responses. In refractory melanoma mouse models, BO-110 synergized with anti-PD-L1 immunotherapy in suppressing tumor growth. Overall, we have characterized new mechanisms of ICB resistance mediated by MDK; yet, we also found that dsRNA-based therapies are able to potentiate ICB responses, even in refractory tumors.El melanoma cutáneo es la forma más letal de cáncer de piel, ya que se caracteriza por tener un alto potencial metastásico y una notable capacidad para evadir el sistema inmunitario. La inmunoterapia usando inhibidores (ej. Anti-PD1/PD-L1, anti-CTLA4) de los “Puntos de Control Inmunitario” (ICB, por sus siglas en inglés) es actualmente la forma más exitosa de tratar el melanoma metastásico. Desafortunadamente, las respuestas clínicas a los ICB siguen siendo limitadas y/o transitorias. La resistencia a los ICB a menudo se observa en pacientes con tumores poco inmunogénicos (tumores fríos) o con alta infiltración de células inmunosupresoras, como macrófagos tumorales (TAMs), células T reguladoras (Tregs) y células mieloides supresoras (MDSCs). Los mecanismos que favorecen el reclutamiento y la modulación de estas células inmunosupresoras -y cómo inactivarlas- son aún interrogantes con un gran impacto clínico para el melanoma. Los objetivos principales de esta Tesis fueron: (i) caracterizar los mecanismos de resistencia a ICB en el melanoma, fijando especial interés en células mieloides supresoras (TAM y MDSCs), e (ii) identificar nuevos agentes terapéuticos capaces de reconvertir las células mieloides en potentes células anti-tumorales. Con este fin, realizamos análisis proteómicos y transcriptómicos para identificar nuevos factores tumorales que pudieran estar implicados en la metástasis y la resistencia a los fármacos. En particular nos enfocamos en MIDKINA (MDK), un factor secretado por células tumorales que promueve la linfangiogénesis y la metástasis del melanoma. Hemos observado que MDK –vía el receptor ALK- activa la vía de NF-κB en células de melanoma, lo que induce un programa secretor pro-inflamatorio que aumenta el reclutamiento de MDSCs y Tregs. Además, observamos que el secretoma inducido por MDK favorece la fosforilación de STAT3 en células mieloides, lo cual promueve su actividad inmunosupresora. Modulando los niveles de expresión de MDK, hemos demostrado que su secreción previene la respuesta terapéutica a los ICB in vivo. Asimismo, hemos observado que los niveles de expresión de MDK correlacionan con una peor respuesta a la terapia anti-PD1 en pacientes con melanoma, lo que demuestra la relevancia de nuestros resultados. En paralelo, hemos observado que nano-partículas de ARN de doble cadena (BO-110) pueden revertir esta inmunosupresión mediante la acción combinada en células de melanoma y macrófagos. Específicamente, hemos demostrado que BO-110 suprime la secreción de MDK e induce una muerte celular inmunogénica en células de melanoma, mientras que en macrófagos promueve su potencial tumoricida, generando una potente inmunidad anti-tumoral. Además, hemos observado que BO-110 potencia la actividad terapéutica de los bloqueantes anti-PDL1 en modelos resistentes de melanoma de ratón. Por lo tanto, BO-110 representa una alternativa terapéutica muy eficiente. En resumen, hemos caracterizado nuevos mecanismos de resistencia a ICB mediados por la proteína MDK; y hemos demostrado que las terapias basadas en ARN de doble cadena son capaces de potenciar las respuestas a ICB, incluso en tumores refractarios.This work has been supported by the following fellowships and grants: - PhD international “la Caixa” Fellowships. 2012-2016. - RETOS Program. María S. Soengas. Spanish Ministry of Economy and Competitiveness. SAF2017-89533-R (2017-2018). - NANODENDMED II-CM. María S. Soengas. COMUNIDAD AUTONOMA DE MADRID. B2017/BMD-3703. 2012-2018. - Asociación Española Contra el Cáncer. María S. Soengas. AECC. 2016-2018

Physiological models for in vivo imaging and targeting the lymphatic system: Nanoparticles and extracellular vesicles.

Imaging of the lymphatic vasculature has gained great attention in various fields, not only because lymphatic vessels act as a key draining system in the body, but also for their implication in autoimmune diseases, organ transplant, inflammation and cancer. Thus, neolymphangiogenesis, or the generation of new lymphatics, is typically an early event in the development of multiple tumor types, particularly in aggressive ones such as malignant melanoma. Still, the understanding of how lymphatic endothelial cells get activated at distal (pre)metastatic niches and their impact on therapy is still unclear. Addressing these questions is of particular interest in the case of immune modulators, because endothelial cells may favor or halt inflammatory processes depending on the cellular context. Therefore, there is great interest in visualizing the lymphatic vasculature in vivo. Here, we review imaging tools and mouse models used to analyze the lymphatic vasculature during tumor progression. We also discuss therapeutic approaches based on nanomedicines to target the lymphatic system and the potential use of extracellular vesicles to track and target sentinel lymph nodes. Finally, we summarize main pre-clinical models developed to visualize the lymphatic vasculature in vivo, discussing their applications with a particular focus in metastatic melanoma.The authors gratefully acknowledge the support of the following sources of funding: M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2017-89533-R), Team Science and Established Investigator awards by the Melanoma Research Alliance, grants from Worldwide Cancer Research and Fundación ‘La Caixa’ Health Research 2019, and a collaborative grant from the Asociación Española Contra el Cáncer (AECC). H.P. acknowledges RETOS SAF2017-82924-R (AEI/10.13039/501100011033/FEDER-UE), Fundación Ramón Areces and La Caixa Foundation (HR-18-00256). We are also grateful for the support of the Translational NeTwork for the CLinical application of Extracellular VesicleS, TeNTaCLES. RED2018-102411-T(AEI/10.13039/501100011033). D.O. is funded by grants from the Spanish Ministry of Health (AES-PIS PI18/1057) and ‘Fundación BBVA-Becas Leonardo a Investigadores y Creadores Culturales 2018’. D.C.-W. was a recipient of a predoctoral fellowship from Fundación ‘La Caixa’ and currently with a Cancer Research Institute Irvington Postdoctoral Fellowship. E.C. is funded by the European Union’s Horizon 2020 research and innovation programme “proEVLifeCycle” under the Marie Skłodowska-Curie grant agreement No 860303.N

A brief overview of cancer research in the elderly population

Cancer is a complex disease and numerous treatments have been proposed and used. The breakthrough has been the use of biological therapy with checkpoint inhibitors, decreasing tumor burden. The life expectancy of cancer patients has increased. However, the major problem refers to the high incidence of tumors in the elderly population most of which have comorbidities.El cáncer es una enfermedad compleja en el cual se han utilizado numerosos tratamientos. El mayor avance en tratamiento ha sido el uso de inhibidores de puntos de control, disminuyendo la carga tumoral. La esperanza de vida de los pacientes con cáncer ha aumentado. Sin embargo, se mantiene la alta incidencia de tumores en la población longeva, la mayoría de los cuales tienen comorbilidades

UNR/CSDE1 drives a post-transcriptional program to promote melanoma invasion and metastasis

RNA binding proteins (RBPs) modulate cancer progression through poorly understood mechanisms. Here we show that the RBP UNR/CSDE1 is overexpressed in melanoma tumors and promotes invasion and metastasis. iCLIP sequencing, RNA sequencing, and ribosome profiling combined with in silico studies unveiled sets of pro-metastatic factors coordinately regulated by UNR as part of RNA regulons. In addition to RNA steady-state levels, UNR was found to control many of its targets at the level of translation elongation/termination. Key pro-oncogenic targets of UNR included VIM and RAC1, as validated by loss- and gain-of-function studies. Our results identify UNR as an oncogenic modulator of melanoma progression, unravel the underlying molecular mechanisms, and identify potential targets for this therapeutically challenging malignancy.L.W. was supported by the Fonds National de la Recherche, Luxembourg, and cofunded by the Marie Curie Actions of the European Commission (FP7-COFUND) (Project Code 1072489). M.G.-F. was supported by a Juan de la Cierva fellowship from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was supported by MINECO and the European Regional Development Fund (ERDF) under grant BFU2012-37135 and BFU2015-68741 to F.G., and Consolider CSD2009-00080 and TV’13-20131430 (Marató de TV3) grants to F.G. and M.S.S. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013–2017, SEV-2012-0208 (to CRG) and SEV-2011-0191 (to CNIO

Involvement of CD38 in the development of CIA.

<p>(A) Cumulative incidence of CIA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033534#s2" target="_blank">Results</a> are expressed as the mean ± SD of the percentage of affected mice at the indicated weeks after immunization. (B) Clinical severity of CIA at different periods after immunization with col II-CFA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033534#s2" target="_blank">Results</a> are expressed as the mean ± SD of the clinical score in each mouse. (C) Paw inflammation was measured 7 weeks after immunization using a digital caliper. The mean values of the inflammation in the four paws of every individual mouse are expressed. Solid bars represent the mean value of each examination. Dotted bars represent the mean ± 3SD of the values found in their respective non-immunized control groups. Statistic differences between WT and CD38 KO mice are indicated as follow: *p<0.05, **p<0.01, *** p<0.001.</p