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The Role of Protein Kinase N in Gastric Cancer

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The Role of Protein Kinase N in Gastric Cancer The Role of Protein Kinase N in Gastric Cancer Lizbeth Minerva Jiménez Flores ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. La consulta de esta tesis queda condicionada a la aceptación de las siguientes condiciones de uso: La difusión de esta tesis por medio del servicio TDR (www.tdx.cat) y a través del Repositorio Digital de la UB (diposit.ub.edu) ha sido autorizada por los titulares de los derechos de propiedad intelectual únicamente para usos privados enmarcados en actividades de investigación y docencia. No se autoriza su reproducción con finalidades de lucro ni su difusión y puesta a disposición desde un sitio ajeno al servicio TDR o al Repositorio Digital de la UB. No se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR o al Repositorio Digital de la UB (framing). Esta reserva de derechos afecta tanto al resumen de presentación de la tesis como a sus contenidos. En la utilización o cita de partes de la tesis es obligado indicar el nombre de la persona autora. WARNING. On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tdx.cat) service and by the UB Digital Repository (diposit.ub.edu) has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized nor its spreading and availability from a site foreign to the TDX service or to the UB Digital Repository. Introducing its content in a window or frame foreign to the TDX service or to the UB Digital Repository is not authorized (framing). Those rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. The Role of Protein Kinase N in Gastric Cancer by Lizbeth Minerva Jiménez Flores A thesis submitted in fulfillment of the requirements for the title of PhD by the University of Barcelona PhD program in Biomedicine The thesis was carried out at Group of Biomedical Research in Digestive Tract Tumors Centre for Molecular Biology and Biochemistry Investigation in Nanomedice (CIBBIM-Nanomedicine) Vall d’Hebron Research Institute Director: Co-Director: Diego Arango del Corro José Higinio Dopeso González PhD candidate: Lizbeth Minerva Jiménez Flores Esta tesis doctoral ha sido apoyada por el gobierno de los Estados Unidos Mexicanos, a través del programa nacional de Becas en el extranjero (convocatoria de 2014 segundo periodo) Registro y Solicitud de Beca No. 269733/383724 Número de Becario 389474 2 A mis padres y a mi hermana 3 Abstract Gastric cancer (GC) is one of the most common types of cancer in the Western World and accounts for over 700,000 deaths every year worldwide. The prognosis is dismal, with an average 5-year survival rate of less than 20%, mainly because of late diagnosis, due to the early stages are clinically silent. The cause of GC is multifactorial, as infectious agents, enviromental or/and genetic factors. Based on Lauren’s histologic classification, there are 2 types of GC: intestinal (IGC) and diffuse (DGC). Diffuse carcinoma cells lacks cohesion and invade tissues independently or in small clusters, is more common in young patients and behaves more aggressively than the intestinal type. Recent findings published in Nature and Nature Genetics identified frequent hotspots (14-24%) mutations in the small GTPase RHOA in the diffuse type of gastric tumors. To investigate the mechanism that underlies downstream of RHOA we analyzed the capacity of the mutations more commonly found in gastric tumors to bind to different effectors. We observed that the mutations found in gastric tumors specifically affect the capacity of RHOA to bind to PKN effector family. Therefore, in this thesis we study and characterized the role of PKN1 in GC. We demonstrated that the downregulation of PKN1 with shRNA or the deletion mediated by CRISPR/Cas9 results in the increase of proliferation in the diffuse gastric cell lines “in vitro” and “in vivo”. Moreover, the opposite effect is observed when we overexpress the constitutively active form of PKN1 in diffuse gastric cell lines with moderate or low levels of PKN1. In addition, we use a novel mouse model with conditional expression in the gastric mucosa of the RHOA-Y42C mutation (the most frequent mutation found in DGC) to investigate the role of RHOA in gastric tumorogenesis initiated by either N-methyl-N-nitrosourea (MNU) or Apc mutations. The mice with expression of the RHOA-Y42C have a significant increase in the number of tumors indicating that RhoA-Y42C is important for progression of the gastric tumors. Finally, we performed a preclinical testing of a new therapeutic approach, such as dietary supplements of arachidonic acid, a well-established activator of PKN1. The work carried out in this thesis will shed light on the newly identified deregulation of RHOA-PKN signaling in gastric cancer and provide a solid rationale for therapeutic targeting of this pathway. 4 Resumen El cáncer gástrico (CG) es uno de los tipos de cáncer más comunes en el mundo occidental y representa más de 700,000 muertes cada año en todo el mundo. El pronóstico es bajo, con una tasa de supervivencia promedio de 5 años de menos del 20%, principalmente debido a un diagnóstico tardío, debido a que las etapas tempranas son clínicamente silenciosas. La causa del CG es multifactorial, como agentes infecciosos, factores ambientales y/o genéticos. Según la clasificación histológica de Lauren, hay 2 tipos de CG: intestinal (CGI) y difuso (CGD). Las células de carcinoma de tipo difuso carecen de cohesión e invaden los tejidos de forma independiente o en pequeños grupos, es más común en pacientes jóvenes y se comporta de forma más agresiva que el tipo intestinal. Hallazgos recientes publicados en Nature and Nature Genetics identificaron mutaciones frecuentes en sitios específicos o hotspots (14-24%) en la proteína GTPasa RHOA en los tumores gástricos de tipo difuso. Para investigar el mecanismo que subyace en la vía de señalización de RHOA, analizamos la capacidad de las mutaciones más recurrentes encontradas en tumores gástricos para unirse a sus diferentes efectores. Observamos que las mutaciones encontradas en los tumores gástricos afectan específicamente la capacidad de RHOA para unirse los efectores de la familia de proteínas kinasas PKN. Por lo tanto, en esta tesis estudiamos y caracterizamos el papel de PKN1 en el CG. Demostramos que la depleción de PKN1 mediante shRNA o la deleción mediada por CRISPR/Cas9 da como resultado el aumento de la proliferación en las líneas celulares gástricas difusas "in vitro" e "in vivo". Además, el efecto opuesto se observa cuando sobreexpresamos la forma constitutivamente activa de PKN1 en líneas celulares gástricas difusas con niveles de expresión moderados o bajos de PKN1. Así mismo, utilizamos un nuevo modelo murino con expresión condicional en la membrana mucosa gástrica de la mutación RHOA-Y42C (la mutación más frecuente encontrada en CGD) para investigar el papel de RHOA en la tumorogénesis gástrica iniciada por N-metil-N-nitrosourea (MNU) o por mutaciones de Apc. Los ratones con expresión de RHOA-Y42C presentaron un aumento significativo en el número de tumores, lo cual indica que RHOA-Y42C tiene un papel importante en la progresión de los tumores gástricos. Finalmente, realizamos una prueba preclínica de un nuevo enfoque terapéutico, como los suplementos dietéticos de ácido araquidónico, un activador bien establecido de PKN1. El trabajo llevado a cabo en esta tesis destacará la importancia de la desregulación recientemente identificada de la señalización RHOA-PKN en el cáncer gástrico y proporcionará una base sólida para la orientación terapéutica de esta vía. 5 INDEX Abstract ....................................................................................................................................... 4 INDEX ......................................................................................................................................... 6 Figure index ........................................................................................................................... 10 Table index ............................................................................................................................ 12 CHAPTER I. Introduction ........................................................................................................... 13 1. The human gastrointestinal system ....................................................................................... 14 1.1 Overview .........................................................................................................................
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