Dissertation committee:
Ιωάννης Τρουγκάκος, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Ισιδώρα Παπασιδέρη, Ομότιμη Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Ανδρέας Σκορίλας, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Ευστάθιος Καστρίτης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Παναγούλα Κόλλια, Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Μαριάννα Αντωνέλου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Μαργαρίτης Αυγέρης, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Organisms are continuously exposed to a plethora of stress-inducing factors and therefore it is essential to ensure the integrity of both their genome and proteome. In particular, in order to maintain the functionality of their proteome, cells have developed the proteostasis network (PN). Key elements of this network include, among others, the two main degradation machineries, namely the Ubiquitin-Proteasome system (UPS) and Autophagy-Lysosome system (ALS), the oxidative stress-related pathway of antioxidant responses that is regulated by NRF2 (Nuclear factor -erythroid derived 2-like 2, NFE2L2)/ KEAP1(Kelch-like ECH-associated protein 1) and the molecular chaperones network. Nevertheless, recent studies by us and others indicate that during ageing both the protein machines and the cellular stress response pathways become dysfunctional as they are deregulated by accumulating damage; these catastrophic events correlate with organismal ageing, cellular senescence and most (if not all) age-related diseases, including cancer. Given that proteostasis imbalance likely represents a hallmark of carcinogenesis, the aim of this study was to analyze the functional role of key (or newly discovered) regulators of the PN and antioxidant response pathways during carcinogenesis and highlight novel anti-cancer approaches and biomarkers with the potential to develop innovative anti-cancer therapies.
The first part of this work focuses on the study of the role of endogenously produced Hydrogen Sulfide in the PN, which, as mentioned above, is disturbed during carcinogenesis. For this purpose, mouse embryonic fibroblasts (MEFs) were isolated from cystathionine γ-lyase (CSE) KO or mercaptopyrostaphylate sulfotransferase (3-MST) KO mice. Our results showed that dysfunction of Hydrogen Sulfide biosynthesis leads to disruption of the PN, as we found a decrease in proteolytic pathway activity and antioxidant responses. The induced disruption of the PN (a milestone of carcinogenesis) indicates the possible involvement of the Hydrogen Sulfide biosynthesis network in carcinogenesis.
The second part of this thesis aimed to investigate the differential response of the proteostasis network between cancer and normal cells during short-term starvation and chemotherapy in breast cancer. Currently, a range of anti-cancer treatments exists; however, they often produce diverse side effects because they indiscriminately affect both normal and cancer cells, lacking sufficient targeting specificity. Short-term starvation has been found to have a positive impact on cancer-free survival. It also appears to sensitize cancer cells to chemotherapeutic agents and reduce chemotherapy-related adverse effects. Our results showed that the combination of fasting and chemotherapy treatment increased the endogenous levels of Reactive Oxygen Species (ROS) and decreased the antioxidant response inducing genomic damage in cancer cells. Notably, this combination did not significantly affect normal cells. Also, our data (along with data from our collaborators in this project) suggests that this effect is mediated, at least in part, through metabolic rewiring and enhanced mitochondrial dysfunctionality in cancer cells.
During the third part of this PhD thesis, we analyzed the expression of PN related genes and miRNAs in both bladder cancer biopsies from patients and in cancer cell lines. Urological carcinoma is one of the most common cancer types worldwide and is considered a disease with high heterogeneity, not only at the molecular level due to the accumulation of a multitude of genetic mutations and epigenetic modifications occurring during disease progression, but also due to variations in prognosis and treatment outcome. Our data showed a significant downregulation of the HSPB2 and HSPB3 genes expression in bladder tumors as compared to matched normal urothelium; yet increased HSPB2 and HSPB3 levels were noted in muscle-invasive (T2–T4) vs. superficial tumors (TaT1), as well as in high-grade vs. low-grade tumors. Survival analyses indicated the significantly higher risk for post-treatment disease relapse in TaT1 patients poorly expressing HSPB2 and HSPB3 genes; this effect tended to be inverted in advanced disease stages (muscle-invasive tumors) where low expression of the HSPB2 and HSPB3 genes delayed disease evolvement. These findings indicate the biphasic impact of HSPB2 and HSPB3 genes in bladder cancer progression. The pro-survival role of HSPB2 and HSPB3 in advanced tumor cells was also evident by our finding that HSPB2, HSPB3 genes expression silencing in high-grade bladder cancer cells enhanced doxorubicin toxicity.
This study also analyzed the biphasic impact of mir-101 in bladder cancer patients' biopsies. Specifically, we analyzed the mechanism of action of miR-101 and its association with proteasome function, using models of multistage carcinogenesis induced either genetically or chemically, along with cancer cell lines expressing or lacking tumor suppressor genes or oncogenes. Our results suggest that oncogenes may trigger mir-101 activation while its expression can be repressed by the tumor suppressor genes p53/p21. Furthermore, we found that mir-101 overexpression increased the chemosensitivity of cancer cells to chemotherapeutic agents and it reduced their migratory potential.
In summary, this study focused on a thorough investigation of the PN role in carcinogenesis. Initially, we found that the PN undergoes significant alterations following Hydrogen Sulfide biosynthesis dysfunction. Additionally, we investigated the differential response of normal vs. cancer cells when exposed to a combination of chemotherapy and fasting. Our data indicate that although PN activation provides protective effects in the early stages of carcinogenesis, in advanced disease stages, it likely significantly contributes to the enhancement of malignant metastatic phenotypes. Finally, an important finding of this PhD thesis is the differential expression of mir-101 during carcinogenesis and its possible mechanism of action.
Keywords:
proteostasis, cancer, chemotherapy, hydrogen sulfide, biomarkers
