Genetic and functional intratumoral heterogeneity in a pancreatic mouse cancer model

RESUMEN: El adenocarcinoma ductal de páncreas (PDAC) es el tipo de cáncer con peor pronóstico debido a su diagnóstico tardío, la diseminación metastásica temprana y a la falta de tratamientos específicos. Esta enfermedad se caracteriza por una extensa heterogeneidad genética y un microambiente tumoral altamente inflamatorio y desmoplástico, lo que contribuye a la agresividad y a la resistencia a tratamientos de esta patología. Con el objetivo de comprender mejor los mecanismos implicados en la progresión y metástasis del cáncer de páncreas, en esta Tesis Doctoral, propusimos caracterizar la dinámica de la heterogeneidad intratumoral en PDAC. Para ello, combinamos la flexibilidad de un modelo de ratón de cáncer de páncreas con la alta sensibilidad de las tecnologías de secuenciación de nueva generación y de célula única, con una estrategia de trazado genético celular, la cual hemos mejorado. A pesar de un origen monoclonal, hemos demostrado la presencia de poblaciones celulares genéticamente distintas dentro de los tumores. Hemos identificado además fenómenos evolutivos de competencia y selección, similares a los descritos en humanos, con la particularidad de que algunos de estos clones presentan una capacidad metastática específica de tejido. Además, hemos descrito que estos tumores suelen presentar alteraciones en Kras y Cdkn2a, pero no en Trp53 o Smad4, al contrario que en humanos. También observamos de manera recurrente la activación de la ruta mutada de Kras, esencial para la generación de metástasis en el hígado, pero no en la de pulmón, que parece ocurrir de manera temprana. Por otra parte, hemos generado una nueva herramienta de trazado genético que puede ser útil para estudiar la heterogeneidad intratumoral. Por último, utilizando las tecnologías de secuenciación de célula única hemos identificado la presencia de distintos grupos transcripcionales en los tumores y metástasis de nuestro modelo, siendo algunos de ellos recurrentes entre muestras y con características que sugieren una colaboración entre distintos grupos de células. Curiosamente, las células metastáticas de pulmón presentan un perfil transcripcional característico, con una transición epitelio mesénquima parcial, una sobre activación de la ruta de EGFR y la producción de citoquinas que probablemente estén implicadas en la preparación del nicho metastático. Este perfil transcripcional está presente en una población minoritaria de los tumores primarios, lo que podría significar que se trata de una población premetastática. Finalmente, la mejora de conocimiento sobre los genes y vías moleculares implicadas en la progresión tumoral y la aparición de metástasis puede traducirse en una mejora significativa del tratamiento de los pacientes de PDAC.ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease due to its late diagnosis, early metastatic widespread, and the lack of effective treatments. PDAC is characterized by extensive genetic intratumor heterogeneity and by a highly inflammatory and desmoplastic tumor microenvironment, which contributes to the aggressiveness and drug resistance of the disease. To understand the mechanisms involved in PDAC progression and metastasis, in this Doctoral Thesis, we proposed to characterize the dynamics of intratumor heterogeneity in PDAC. To do this, we combined the flexibility of a well-characterized pancreatic mouse model, the high sensitivity of next-generation and single-cell sequencing technologies, and a multi-fluorescent protein lineage tracing model. First, we have demonstrated the monofocal origin of the aggressive pancreatic tumors from our mouse model and the presence of genetically distinct cell populations within tumors. We have also identified evolutionary phenomena of competition and selection, similar to those described in humans, with the particularity that some of these clones have tissue-specific metastatic capacity. In addition, we have described that these tumors recurrently present amplification of the mutant Kras signal which seems important for the generation of metastases in the liver, but is disposable for the metastasis in the lung, which seems to be the result of early colonization. Finally, we have identified the presence of different transcriptional groups in the tumors and metastases of our model, some of them being recurrent between samples and with characteristics that demonstrate a collaboration between different groups of cells. Interestingly, metastatic lung cells present a particular transcriptional profile, with a partial epithelial-to-mesenchymal transition, overactivation of the EGFR pathway, and the production of cytokines that are probably involved in the preparation of the metastatic niche. This transcriptional profile is present in a minority population of primary tumors, which could mean that it is a premetastatic population. Finally, the improvement of the knowledge about the genes and molecular pathways involved in tumor progression and the appearance of metastasis could be finally translated to a significant improvement in the treatment of PDAC patients.Esta tesis ha sido financiada a través de fondos procedentes de: - Fundación Ramón Areces, Proyecto: “Caracterización molecular del papel de la disfunción mitocondrial en el desarrollo tumoral”. Proyectos para la Ciencia de la Vida y a Materia (2014). - European Research Council, Proyecto: “Molecular characterization of the role of intratumor heterogeneity in cancer progression and metastasis”. ERC-2014-StG-2014-637904. Para la realización de este trabajo, Laura Quevedo también ha recibido: - Beca predoctoral de la Universidad de Cantabria (convocatoria: 2018- 2022). - Ayuda de movilidad predoctoral de la Universidad de Cantabria para realizar una estancia breve en la Technical University of Munich (convocatoria: 2021)

ARID2 deficiency promotes tumor progression and is associated with higher sensitivity to chemotherapy in lung cancer

The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.Financial Support: I. V. is supported by SAF2012-31627 and SAF2016-76758-R grants from the Spanish Ministerio de Economía y Competitividad (MINECO), by a Fundación Ramón Areces grant and by ERC2014-StG637904 grant from the European Research Council. I. V has been awardee of the Programa Ramón y Cajal (MINECO, Spain). T. M has been awardee of the Ayudas para la contratación de investigadores predoctorales (MINECO, Spain). B. M is awardee of the Ayudas para la formación de profesorado universitario (FPU, Ministerio de Educación y Formación Profesional, Spain). PC laboratory is supported by grant SAF-2015-63638R (MINECO/FEDER, UE); by Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and by Asociación Española Contra el Cáncer (AECC), grant GCB141423113. BC has been supported by a Retos Jóvenes Investigadores grant SAF2015-73364-JIN (AEI/FEDER, UE) and a grant from Fundación Francisco Cobos. P. S. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001152), the UK Medical Research Council (FC001152). HUCA/IUOPA which is jointly financed by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III and Fundación Bancaria Cajastur. This research was funded in part by the Wellcome Trust [FC001152]

pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation

The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.Acknowledgements: The authors thank VHIO Proteomics, Molecular Oncology and Genomics Core Facilities for technical assistance. We are grateful to Manuel Serrano for providing several reagents, advice and critical discussion on the manuscript. We also thank Alonso García and Raquel Pérez for their help in processing and analyzing digital images, Gemma Serra and Sandra Peiró for their assistance with subcellular fractionation and immunoprecipitation experiments, Sara Arce and Joaquín Mateo for providing several reagents during the development of critical experiments of this manuscript, and Juan Angel Recio for his help with the cSCC cohort. We are immensely grateful to all the members of the Abad lab for generating the know-how for the identification of novel sORFs, for the critical reading on the manuscript and in general for their constant support to this project. Work in the Abad lab is supported by VHIO, Fero Foundation, La Caixa Foundation, Asociación Española Contra el Cancer (AECC), La Mutua Foundation and by grants from the Spanish Ministry of Science and Innovation (SAF2015-69413-R; RTI2018-102046-B-I00). M.A. was recipient of a Ramón y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2013-14747). O.B. is recipient of a FPIAGAUR fellowship from Generalitat de Catalunya. We also acknowledge funding from grant PGC2018-094091-B-I00 from the Spanish Government

Development of new cell genetic tracing tools for the study of intratumour heterogeneity

Intratumour heterogeneity has been observed in multiple cancers and has been postulated as a critical aspect for tumour metastasis and treatment resistance. Therefore, a further characterization of its role in cancer progression and metastasis has become essential to increase our understanding of cancer biology and to improve the treatment of cancer patients. The use of cell lineage tracing systems, combined with the use of genetically modified mouse models, could be applied in this context. Several genetic tracing systems, based in a random CRE-mediated recombination event in an allele with multiple fluorescent markers surrounded by incompatible lox sites have been created. Once this recombination occurs, the cell and its genetic descendants are permanently labelled with the same fluorescent marker. Nevertheless, these systems present several limitations like a reduced number of potential colour combinations or problems in the unique identification of the markers. Here we have identified new incompatible lox sites that, together with an efficient selection of fluorescent markers, have allowed us to design a new system that will be able to produce up to 15 different colour combinations that can be uniquely identified by confocal microscopy and FACS. This system will be combined with cancer mouse models to study the role and dynamics of intratumour heterogeneity in cancer progression.Máster en Biología Molecular y Biomedicin

Política criminal y “prevención”

Este libro es el producto de las investigaciones de 2014 que se socializaron en el Congreso Nacional de Política Criminal y "Prevención" en el que se presentaron diferentes posturas y críticas al concepto de "prevención" del delito. La primera parte del libro la hemos denominado "Una crítica al concepto de prevención", en ella se contemplan los siguientes capítulos: "Políticas públicas y 'prevención' en Colombia", en este se confronta una política criminal garantista con una política criminal reactiva; "Prevenciones sobre la prevención: algunas consideraciones desde la criminología", allí se analizan ciertos argumentos clásicos de las políticas de prevención del delito, señala algunos de sus límites y elabora algunas críticas contemporáneas; "Una política criminal desde la garantía de los derechos económicos sociales y culturales: una aproximación al enfoque de género", identifica algunos obstáculos para el logro de una política de prevención del delito desde la garantía de los derechos económicos, sociales y culturales en las mujeres; "La necesidad de una política preventiva verde en Colombia", en el que se desarrolla la tesis según la cual una macropolítica pública de prevención de daños en Colombia debe tener como constituyente central el componente ambiental; y "Programas socioeducativos para resocialización en el contexto penitenciario", este analiza la efectividad de cinco programas socioeducativos en prisión. La segunda parte del libro se titula "Algunos métodos para una política criminal preventiva", en el que encontramos los siguientes capítulos: "Propuesta metodológica para el análisis jurídico-económico del delito: construcción de indicadores auxiliares en la toma de decisiones de política criminal", que propone una metodología para el análisis jurídico-económico del delito; y "Métodos alternativos de solución de conflictos en la política criminal del Estado", el cual aborda la eficacia de la implementación y aplicación obligatoria de la conciliación

PLCy1/PKC0 Downstream Signaling Controls Cutaneous T-Cell Lymphoma Development and Progression

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLC?1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLC?1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLC?1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKC?) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKC?-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKC? blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKC? target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKC?) in 81 human mycosis fungoides samples. In summary, PKC? can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.ACKNOWLEDGMENTS: This work has been funded by the Instituto de Salud Carlos III (ISCIII)/FEDER (PI16/00156, PI19/00204, and ASOCIACION LUCHAMOS POR LA VIDA to JPV; PI17/0957 to PLOR). NGD has been supported by a predoctoral contract from UC-IDIVAL. BC holds a RyC contract from MICINN (RYC2018-024004). AEC is funded by ISCIII/MINECO/FEDER (PT17/0009/0019)

Libro de Proyectos Finales 2021 primer semestre

PregradoIngeniero CivilIngeniero de SistemasIngeniero ElectricistaIngeniero ElectrónicoIngeniero IndustrialIngeniero Mecánic