Estudio de la familia de proteínas quinasas C como nodos de control en la señalización celular

Abstract

This PhD Thesis has deepened the knowledge of the function of different isoforms of the PKC family, such as PKCα and PKCε, through the study of their signaling in different cellular processes. During this PhD Thesis, PKCα-mediated signaling has been studied and characterized in two specific subtypes of breast cancer: Luminal A subtype, using MCF-7 cells as a model; and the basal or triple negative subtype, where MDA-MB-231 cells were used as a cellular model. To understand how PKCα signaling was involved in the regulation and development of the tumour process in MDA-MB-231 cells, a cell line that stably inhibited PKCα expression was developed by the gene editing tool CRISPR-Cas9. Through the morphological and functional study of this model cell line, and its comparison with the wild-type line, it has been possible to identify how the lack of PKCα and its signaling causes morphological alterations in the cells, mainly affecting the cytoskeleton and its three-dimensional organization. In addition, PKCα was identified as an important kinase in tumor processes such as cell migration or invasion, being seriously affected. To further investigate how PKCα causes these alterations in the morphology and functionality of MDA-MB-231 cells, a comparative proteomic study was performed between both cellular lines, where the activation state of the cellular kinome after PKCα inhibition was analyzed, as well as the proteome and phosphoproteome. The integration of this information allowed the identification of PKCα as an important element for the correct functioning of the cell cycle, as well as its role in the regulation of the cytoskeleton and cell motility processes through the regulation of the expression and/or activity of structural proteins and essential components such as small GTPases. This information was used for the development of a combination therapy, where PKCα inhibition acts as a central element, thus identifying the oncogenic role of the isoform in triple-negative breast cancer. Similarly, the study of the signaling pathways where PKCα appears involved in MCF-7 cells, through its inhibition by small interfering RNA, was carried out. The information collected from those proteins that experienced alterations in their expression after PKCα inhibition, added to the identification of the activation state of different kinases through the kinase array, showed PKCα as a key kinase in the regulation of numerous pathways necessary for tumor development. For example, the MAP kinase signaling pathway, PI3K-AKT, interferon signaling or p53, were affected after PKCα inhibition. This information, added to the functional alteration experienced by MCF-7 cells, places PKCα as an oncogenic kinase in this mammary tumor subtype. Finally, we studied how PKCε participates in cell degranulation processes in RBL-2H3 cells. Phosphatidic acid is an important membrane phospholipid involved in many functions and signaling events. However, how its regulation affects PKCε functionality in these cells was not known. The results obtained using TIRF microscopy revealed how this signaling would exert a role in stabilizing and preparing vesicles for vesicle fusion processes through phosphorylation phenomena on SNAP23, thus preparing them for possible calcium stimuli leading to degranulation of RBL-2H3 cells. These results place PKCε as a possible target in allergic reactions or reactions triggered by mast cell activation.