Dissertation committee:
1) Μαρία Ρουμπελάκη, Επίκουρος Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
2) Ευάγγελος Μαρίνος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
3) Παναγιώτης Πολίτης, Ερευνητής Γ΄, ΙΙΒΕΑΑ
4) Αριστείδης Ηλιόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
5) Μαρία Γαζούλη, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
6) Αθανάσιος Κοτσίνας, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
7) Αριστείδης Χαρώνης, Ερευνητής Α΄, ΙΙΒΕΑΑ
Summary:
Within the last years, systems medicine has emerged as a holistic approach to tackle complex diseases, such as cancer and cardiovascular disease. High-throughput omics approaches are integral part of systems approaches and among them proteomics has a central role. Tissue proteomics may provide a wealth of information on the molecular mechanisms of such complex diseases thus contributing to the identification of novel druggable targets for therapeutic interventions.
Bladder cancer is the most costly and second in mortality among urogenital cancers. Patients with advanced stages of bladder cancer are characterized by poor outcome which can be partially explained by the limited therapeutic options. This indicates an imperative need for the identification of novel druggable targets. To this end, this study has focused on the characterization of the global proteomic changes underlying bladder cancer invasion at the tissue level. By performing a comprehensive proteomic analysis, 144 proteins were found to be significantly differentially expressed between muscle invasive and non-muscle invasive bladder cancer tissues. Several of these proteins had been previously reported in the context of bladder cancer, such as annexins, alpha actinins, TGFBI, cadherins and cathepsins, increasing the validity of this approach. Remarkably, this study also revealed numerous novel findings such as the differential expression of FUCA1, BROX, PGRMC1, and PSMD12 in invasive bladder cancer which was further confirmed by immunohistochemistry. In silico analysis of the differentially expressed proteins predicted, among others, a significant activation of protein synthesis. EIF3D and RHEB, both associated with translation, were prioritized for functional analysis in vitro and in vivo. Knock-down of EIF3D in the metastatic bladder cancer cell line T24M, inhibited cell proliferation, migration and colony formation in vitro and consequently led to a decrease of tumor growth in vivo. Similar biological effect was observed upon the knock-down of RHEB in vitro yet not in vivo. Therefore, in this study, EIF3D was highlighted as a novel candidate involved in bladder cancer invasion having also a therapeutic potential.
On the other hand, cardiovascular disease is the leading cause of death worldwide with a high mortality rate and an increasing incidence annually. Atherosclerosis, the major cause of cardiovascular disease, is a chronic, systemic disease with a long-lasting subclinical phase. Early detection and treatment of patients with atherosclerosis are crucial in order to reduce the burden of the disease. To this end, and specifically aiming to gain insight into the early cardiovascular disease mechanisms, in this study, a systematic approach based on mouse models, human tissues and in vitro studies, was applied. Proteome analysis using high-resolution tandem mass spectrometry (LC-MS/MS) of thoracic aortas from atherosclerotic mice models, Ldlr-/- and ApoE-/-, in the presence or absence of diabetes was performed. One hundred seventy-seven proteins were found to be differentially expressed in all models in comparison to wild type (WT) animals, suggesting that these changes were associated with atherosclerosis, regardless of the molecular background of the model used. These proteins segregated into atherosclerosis relevant processes including metabolism, hemostasis, immune system, extracellular matrix and chromatin organization. Further LC-MS/MS experiments using human vessels from patients with cardiovascular disease and controls, confirmed the upregulation of six proteins that were identified also in mice. Among them, KDM5D, an epigenetic modulator – member of the KDM5 family of histone demethylases, exhibited pronounced overexpression in disease. Along the same lines, a reduction of the trimethylated form of H3K4, which is the substrate of KDM5 demethylases, was detected in the same samples using Western blot analyses. Further functional interference studies showed an impact of KDM5 inhibition on human endothelial (HUVEC) cells, including a reduction of cell proliferation, migration and tube-forming ability in vitro. In conclusion, the data of this study support a role for KDM5 histone demethylases in the development of cardiovascular disease, likely via affecting H3K4 methylation. The proteomics findings of this study hold the promise to further support systems biology investigations in the future.
Keywords:
Bladder cancer, Metastasis, Cardiovascular disease, Atherosclerosis, Proteomics
