Mecanismos moleculares de desregulación de la proteína SET y estudio de su valor pronóstico y terapéutico en cáncer colorrectal

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 24-05-2019El cáncer colorrectal (CCR) es la tercera patología tumoral de mayor incidencia y la cuarta causa de muerte más común por cáncer en el mundo. La proteína fosfatasa 2A (PP2A) es una serina/treonina fosfatasa que actúa como supresor tumoral regulando un gran número de rutas de señalización de gran relevancia en cáncer, y su desregulación supone un evento crucial en el proceso de transformación maligna. La proteína SET es un potente inhibidor endógeno de PP2A, que se ha descrito frecuentemente desregulado en CCR metastásico (CCRm), donde determina sensibilidad a distintos tratamientos estándar. Así, la sobreexpresión de SET se asocia con una menor supervivencia global (SG) y una menor respuesta a la quimioterapia basada en oxaliplatino (LOHP) y 5-fluorouracilo (5- FU). Este proyecto de investigación se centra en el estudio de los mecanismos moleculares que contribuyen a la desregulación de SET así como en determinar su impacto clínico en pacientes con CCR localizado. Para ello, se han llevado a cabo una gran variedad de ensayos moleculares para evaluar la importancia biológica y funcional de la desregulación de SET, los cuales se describirán en detalle en el contenido de esta memoria. La tesis doctoral se presenta como un compendio de publicaciones, la primera de las cuales muestra que la expresión de SET y el microRNA-199b (miR-199b), regulador negativo de esta proteína, están inversamente correlacionadas. Así, la sobreexpresión de este miR impide la proliferación celular y aumenta la respuesta a LOHP debido a la activación de PP2A a través de la inhibición de SET. Además, demostramos en este trabajo que los casos con una baja expresión del miR-199b presentan menor SG. En el segundo artículo observamos alta expresión de SETBP1, regulador positivo de SET, en varias líneas celulares de CCR pero con prevalencia muy baja en pacientes, por lo que estudiamos la existencia de mutaciones de SETBP1 como mecanismo alternativo a su sobreexpresión. Sin embargo, no encontramos mutaciones en este gen, por lo que de existir constituyen eventos poco frecuentes en CCR. En el tercer trabajo se analizó la expresión de caveolina 1 (CAV-1) en CCR localizado, proteína relacionada tanto con inhibición de PP2A como con Rac-1, mediador clave en la migración inducida por SET. La baja expresión encontrada en el 76.2% de los casos sugiere que CAV-1 podría actuar como supresor tumoral en estadíos tempranos de esta enfermedad. Por último, en el cuarto artículo mostramos que la desregulación de SET se asocia con progresión tumoral y predice un pronóstico adverso en pacientes con CCR localizado. Además, SET regula migración, crecimiento con independencia de anclaje, transición epitelio-mesénquima (TEM) y permite definir a un subgrupo de pacientes de mal pronóstico que podrían beneficiarse del uso de antagonistas de SET en los protocolos clínicos.Colorectal cancer (CRC) is the third most incidence tumor pathology and the fourth common cause of death related with cancer all over the world. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that acts as a tumor suppressor regulating many signaling pathways with high relevance in human cancer, and its deregulation is a crucial event in cancer development. SET protein is a potent endogenous PP2A inhibitor that has been described frequently deregulated in metastatic CRC, where it plays an important role in determining sensitivity to different standard treatments. Thus, SET overexpression is associated with a lower overall survival (OS) and a less response to chemotherapy based on oxaliplatin or 5-fluorouracil (5-FU). This research project focuses on the study of the molecular mechanisms that contribute to SET deregulation as well as to determine its clinical impact in patients with localized CRC. For that, a wide variety of molecular assays have been carried out to evaluate the biological and functional importance of SET deregulation, which will be described in detail in the contents of this report. The doctoral thesis is presented as a compendium of publications, first of which shows that the expression of SET and the miR-199b, a negative regulator of this protein, are inversely correlated. Thus, overexpression of this miR impairs cell proliferation and increases the response to oxaliplatin treatment due to PP2A activation through SET inhibition. Moreover, we demonstrated in this work that those cases with a low expression of miR-199b present a lower OS. In the second article we observed a high expression of SETBP1, positive regulator of SET, in several CRC cell lines but only in two of the patients of our cohort, so we studied the possible existence of mutations in SETBP1 gene as an alternative mechanism to their overexpression. However, we did not found SETBP1 mutations in any of the CRC patients samples included in the study so this alterations are, if exist, rare events in CRC patients. In the third work we focused on analyzing the expression of CAV-1 in localized CRC, since it has been described that this protein is related to both PP2A inhibition and Rac-1, a key regulator in the migration induces by SET. The low expression found in the 76.2% of cases suggests that CAV-1 could act as a tumor suppressor in early stages of this disease. Finally, we showed in the fourth article that SET deregulation is associated with tumor progression and predicts an adverse prognosis in patients with localized CRC. We have also observed that SET regulates cell migration, anchorage-independent cell growth, epithelial to mesenchymal transition (EMT) and also defines a subgroup of patients with poor prognosis that could benefit from the use of SET antagonists in clinical protocols

Downregulation of microRNA-199b predicts unfavorable prognosis and emerges as a novel therapeutic target which contributes to PP2A inhibition in metastatic colorectal cancer

The tumor suppressor microRNA-199b (miR-199b) is a negative SET regulator associated with poor outcome in some human cancers. However, its expression levels as well as potential biological and clinical significance in colorectal cancer (CRC) remain completely unexplored. The PP2A inhibitor SET has shown promising therapeutic and clinical implications in metastatic CRC (mCRC) but the molecular mechanisms underlying SET deregulation are currently unknown. We show here miR-199b downregulation in 4 out of 5 CRC SET-overexpressing cell lines and its inverse correlation with SET overexpression in CRC patients. Moreover, miR-199b led to PP2A activation through a direct SET inhibition, impaired cell viability and enhanced oxaliplatin sensitivity in CRC cells. MiR-199b was found downregulated in 25% of cases, and associated with lymph metastasis (p = 0.049), presence of synchronous metastasis at diagnosis (p = 0.026) and SET overexpression (p < 0.001). Furthermore, low miR-199b levels determined shorter overall (p < 0.001), progression-free survival (p = 0.003) and predicted clinical benefit to oxaliplatin treatment. The miR-199b prognostic impact was particularly evident in both younger and KRAS wild-type subgroups. Multivariate analyses confirmed its independent prognostic impact. Altogether, our results show that miR-199b is a tumor suppressor whose downregulation independently determines worse outcome and emerges as a potential contributing mechanism to inhibit PP2A via SET overexpression in a subgroup of mCRC patient

Microrna-199b downregulation confers resistance to 5-fluorouracil treatment and predicts poor outcome and response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients

Neoadjuvant 5-fluorouracil (5-FU)-based chemoradiotherapy followed by mesorectal excision is the current standard treatment in locally advanced rectal cancer (LARC) and the lack of complete response represents a major problem that compromises long-term patient survival. However, there is a lack of robust established markers predictive of response to this preoperative treatment available in the clinical routine. The tumor suppressor microRNA (miR)-199b directly targets the PP2A inhibitor SET, which has been involved in 5-FU resistance, and its downregulation has been found to correlate with poor outcome in metastatic colorectal cancer. Here, we studied the functional effects of miR-199b on 5-FU sensitivity after its ectopic modulation, and its expression was quantified by real-time-PCR in a cohort of 110 LARC patients to evaluate its potential clinical significance. Interestingly, our findings demonstrate that miR-199b enhances the sensitivity of colorectal cancer cells to 5-FU in a SET-dependent manner, and that both miR-199b overexpression and SET inhibition are able to overcome resistance to this drug using an acquired 5-FU-resistant model. MiR-199b was found downregulated in 26.4% of cases and was associated with positive lymph node levels after chemoradiotherapy (CRT, p = 0.007) and high pathological stage (p = 0.029). Moreover, miR-199b downregulation determined shorter overall (p = 0.003) and event-free survival (p = 0.005), and was an independent predictor of poor response to preoperative CRT (p = 0.004). In conclusion, our findings highlight the clinical impact of miR-199b downregulation predicting poor outcome and pathological response in LARC, and suggest the miR-199b/SET signaling axis as a novel molecular target to prevent the development of resistance to 5-FU treatment.This research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

La armada de Chile y su participación en la misión de estabilización de las Naciones Unidas en Haití (Minustah), historia y evolución

This article aims to analyze the rules that govern the Chilean forces in peace operations, specifying the most relevant characteristics of peacekeeping missions, in addition, to assess the dimensions and components of the Navy participation in Haiti, as the defense of the homeland, has been conceived as the main function of the Armed Forces. In this sence, it�s the Navy�s duty to maintain the security of the country in �times of peace and war�. That is why it has developed the strategy of the three vectors, projecting its work in the areas of Defence, Maritime and International. In this last issue, the Navy has an important role in peacekeeping missions, establishing a regional commitment, especially in their participation in the Stabilization Mission of the United Nations in Haiti.El presente artículo tiene por objetivos analizar las normas que rigen a las fuerzas chilenas en Operaciones de Paz, especificando las características más relevantes de las Misiones de Paz, además, de evaluar dimensiones y componentes de la participación de la Armada en Haití, por cuanto la defensa de la Patria, ha sido concebida como la principal función de las Fuerzas Armadas. En dicha línea, a la Armada le corresponde mantener la seguridad del país en �tiempos de paz y de guerra�. Por lo que, ha desarrollado la estrategia de los Tres Vectores, bajo los que proyecta su quehacer en los ámbitos de Defensa, Marítimo e Internacional. En este último, la Armada tiene un importante rol en Misiones de Paz, estableciendo un compromiso regional, especialmente en su participación en la Misión de Estabilización de las Naciones Unidas en Haití

Diseño de instalación fotovoltaica de 200kW para autoconsumo en una empresa del sector agroalimentario situada en Quart de Poblet

[ES] En este Trabajo Final de Grado se desarrolla el proyecto de una instalación fotovoltaica de 200 kW dedicada principalmente al autoconsumo, aunque también estará conectada a la red de distribución y se podrá verter los excedentes de energía generada, recibiendo una retribución por ello. La instalación se ubica en la cubierta de una nave de una industria agroalimentaria situada en Quart de Poblet, Valencia.[EN] In this Final Degree Project the project of a 200 kW photovoltaic installation dedicated mainly to self-consumption is developed, although it will also be connected to the distribution network and it will be possible to discharge the surplus generated energy, receiving a retribution for it. The installation is located on the deck of a warehouse of an agrifood industry located in Quart de Poblet, Valencia.Torrejón Marín, B. (2018). Diseño de instalación fotovoltaica de 200kW para autoconsumo en una empresa del sector agroalimentario situada en Quart de Poblet. http://hdl.handle.net/10251/10706

VISIÓN MARÍTIMA DE CHILE

This paper seeks to highlight the Maritime Vision of Chile, from the nineteenth century to the twenty-first century; considering the geography and the historical development of our country, outlining the ocean-policy perspective of visionaries, and the current maritime policy of the Chilean state. To achieve this, it�s proposed the following question research: Which was the origin, development and prospects, of our leaders in relation to the maritime vision of the Chilean State?. As a thesis, it�s proposed that 'Chile shows its ocean territory growth and development like a country, in ocean territory country', because the will be a great state would be adjusted to the actual geographical sea, with the skills and human potential, and will that have of our leaders, in the formulation of policies of government, always focused on the national interest. The working hypothesis is correlational in nature, 'the geography and maritime history of our leaders, are directly related to the maritime vision of Chile'.El presente ensayo pretende dar a conocer la Visión Marítima de Chile, desde el siglo XIX al XXI, considerando tanto la configuración geográfica como el desarrollo histórico de nuestro país, esbozando la perspectiva océano-política de hombres visionarios, y la actual política marítima del Estado de Chile. Para lograr lo anterior, se planteó la siguiente pregunta de investigación ¿cuál ha sido el origen, el desarrollo y las perspectivas de nuestros gobernantes y líderes en relación con el pensamiento y visión marítima del Estado de Chile?. Como tesis se plantea que: �Chile demuestra el crecimiento y desarrollo país en su territorio oceánico�, por cuanto, la voluntad de ser un gran Estado estaría acorde con la realidad geográfica marítima, con las capacidades y potencialidades humanas y, por la voluntad que han de nuestros gobernantes, en el planteamiento de sus políticas de gobierno, enfocadas siempre en el interés nacional. La hipótesis de trabajo es de carácter correlacional: �la configuración geográfica y vocación marítima de nuestros gobernantes, están directamente relacionados con la visión marítima de Chile�

La integración curricular como metodología de aprendizaje significativo

The essay on ‘The Curricular Integration and Meaningful Learning Methodology’ is intended to share a classroom experience, whose goal was to promote a participatory inter-chairs, where students and the students, of the Naval Arturo Prat, were capable of turning their efforts in an interactive manner designed to capture, through various communication resources seen in the Department of Spanish Language related to the textual production planning and organizing the information in a consistent and cohesive, respecting the structure addressed, according with various speech acts, together with the development of written texts from a plan, expressing his ideas, according to a specific structure to present work interactively with the support of persuasive resources,highlighting the organization and creativity, showing the values of the school, selected for the activity. In the Department of History and Geography, the objective was to determine the economic significance of our sea, together with the assessment of the political, economic and cultural Chile established the states within the Pacific Rim, to create a maritime awareness the light of the values of the Chilean NavyEl ensayo sobre ‘La Integración Curricular como Metodología de Aprendizaje Significativo’ tiene por finalidad compartir una experiencia de aula, cuyo objetivo fue promover una instancia de participación ínter-cátedras, en donde los alumnos y las alumnas, de la Escuela Naval Arturo Prat, fueron capaces de volcar sus esfuerzos de una manera interactiva, destinada a plasmar, mediante diversos recursos comunicativos vistos en la Cátedra de Lengua Castellana, relacionados con la planificación y producción de textual, organizando la información de manera coherente y cohesionada, respetando la estructura abordada, de acuerdo con los diversos actos de habla, junto con la elaboración de textos escritos a partir de un plan, expresando sus ideas,de acuerdo con una estructura específica, para presentar un trabajo de manera interactiva, con el apoyo de recursos persuasivos. Destaca, a la vez, la organización y creatividad, evidenciando los valores de la Escuela, seleccionados para la actividad. En la Cátedra de Historia y Geografía, el objetivo fue determinar la importancia económica de nuestro mar, junto con la evaluación de las relaciones políticas, económicas y culturales que establece Chile con los Estados integrantes de la Cuencadel Pacífico, para crear una conciencia marítima a la luz de los valores propios de la Armada de Chil

Activation of the Tumor Suppressor PP2A Emerges as a Potential Therapeutic Strategy for Treating Prostate Cancer

Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients