Identification of ISGylated proteins in pancreatic cancer stem cells

• Autores: Laura Martín Hijano
• Directores de la Tesis: Bruno Sainz Anding (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2022
• Idioma: inglés
• Número de páginas: 145
• Títulos paralelos:
  • Identificación y caracterización de proteínas ISGiladas en células madre de cáncer de páncreas
• Tribunal Calificador de la Tesis: Francisco Portillo Pérez (presid.), Mireia Vallespinós Serrano (secret.), Raúl Torres Ruiz (voc.), Laura García Bermejo (voc.), María Elena Martín Palma (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
      • Proteínas
  • Ciencias de la vida
    • Biología celular
  • Ciencias médicas
    • Patología
      • Oncología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most aggressive solid malignancies, with a 5-year survival rate of around 5-7%. PDAC is characterized by a highly metastatic and chemoresistant profile, and with a poor response to current standard therapies. This is believed to be due, at least in part, to a subpopulation of tumor cells known as cancer stem cells (CSCs) endowed with stem-like properties including tumorigenesis, metastatic potential, chemoresistance and relapse. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier (UBL) that conjugates to target proteins, in a stepwise manner with the aid of three ligases (E1, E2 and E3), to post-translationally modify them in a process known as ISGylation. Upregulation of ISG15 and ISGylation has been linked to tumor progression and, more importantly, to promotion and maintenance of cancer stem-like properties across different tumor entities. Previous work has demonstrated the strong connection that exists between the upregulation of ISGylation and the promotion of CSC-properties in PDAC. However, very few ISGylated proteins have been identified and characterized to date, even more so in relation with cancer. To our knowledge, no studies have been conducted to identify ISGylated proteins specifically in pancreatic CSCs (PaCSCs). Hence, in this thesis work a mass spectrometry (MS)- based study has been conducted to identify differentially ISGylated proteins in PaCSCs, aiming to discover potential targets for PaCSC elimination. To do so, we developed two straight-forward methodologies: the first one based on overexpressing the ISGylating machinery (E1, E2 and E3 ligases), including a tagged version of ISG15 (V5-ISG15) and the second one was based on replacing the endogenous ISG15 gene by a tagged version of ISG15 (V5-ISG15) in PDAC cells. Both strategies converge in the differential analysis of V5-ISG15-conjugated proteins in PaCSCs versus non-PaCSCs via Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS). Unfortunately, in the present work we have been unable to reliably identify ISG15-conjugation targets due to technical issues, described and detailed in this thesis. Despite this, our goal is to further improve the technical aspect of this project in order to identify trustworthy ISGylated proteins in PaCSCs and to open diverse research lines based on the discovery of these targets