Identification of ISGylated proteins in pancreatic cancer stem cells
Title (trans.)
Identificación y caracterización de proteínas ISGiladas en células madre de cáncer de páncreasAuthor
Martín Hijano, LauraAdvisor
Sáinz Anding, BrunoEntity
UAM. Departamento de Bioquímica; Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM)Date
2022-05-20Subjects
Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 20-05-2022Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
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
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