Dissertation committee:
Βασίλειος Γοργούλης, Καθηγητής, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Νικόλαος Ανάγνου, Καθηγητής, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Δημήτριος Θάνος, Ερευνητής Α', Ίδρυμα Ιατροβιολογικών Ερευνών της Ακαδημίας Αθηνών
Ανδρέας Σκορίλας, Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Απόστολος Κλινάκης, Ερευνητής Β', Ίδρυμα Ιατροβιολογικών Ερευνών της Ακαδημίας Αθηνών
Αθανάσιος Κοτσίνας, Επίκουρος Καθηγητής, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Μαρία Ρουμπελάκη, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
The Notch signaling pathway is evolutionarily conserved and present in all Metazoans and plays a crucial role in embryonic development and tissue homeostasis. Specifically, it controls cell fate decisions through interactions between neighboring cells by positively or negatively affecting the processes of proliferation, differentiation and apoptosis in a context-dependent manner. Therefore, dysregulation or loss of Notch signaling leads to a wide spectrum of human disorders, from developmental syndromes to cancer, where it has been implicated both as an oncogene and a tumor suppressor.
An initial observation that mice lacking Nicastrin—which leads to Notch pathway inactivation—develop ureteral tumors led to the hypothesis that Notch signaling acts as a tumor suppressor in the urinary tract. First, we showed that Notch pathway is active in normal mouse urothelium. Next, the characterization of the tumors revealed that they are high grade urothelial carcinomas expressing basal cell markers. Therefore, they are classified in the basal subtype which is associated with poor prognosis in humans. Re-expression of the intracellular domain of Notch1 receptor suppresses tumor onset, underlying the fact that Notch signaling loss is a “single hit” driver tumorigenic event.
In a following step, we generated a urothelial-specific transgenic line expressing recombinase Cre, and also the green fluorescent protein, under the transcriptional control of the UroplakinII gene promoter (UpII-Cre-iresGFP). Notch pathway inactivation in the mouse bladder leads to the development of tumors that consist of simple and nodular hyperplasia, as well as high-grade dysplasia and carcinoma in situ showing basal characteristics. In addition, we studied the effect of the loss of each Notch receptor individually and we found that Notch1 and Notch2 are the main mediators of the pathway’s tumor suppressor role in the urothelium. In particular, Notch1 or Notch2 loss induces the phenotype of keratinizing squamous metaplasia in mice introduced in a chemical carcinogenesis protocol. Furthermore, the study of the interaction between Notch pathway inactivation and Kras or Pi3k activation in mice offered some indications towards a synergistic action.
As expected, soon arose the question whether these findings in mice can be translated to humans. First, we observed that Notch pathway is active in normal human urothelium. Subsequently, we showed that Notch1-3 overexpression suppresses the growth of human bladder cancer cell lines. Our next goal was the elucidation of the molecular mechanism of Notch action in the urothelium. We indicated that Notch pathway activation regulates ERK1/2 phosphorylation through direct transcriptional activation of DUSPs and thereby restrains the proliferative output of the MAPK pathway. Molecular characterization of mouse ureter tumors corroborated the identified mechanism.
Surprisingly, we identified novel somatic loss-of-function mutations in Notch pathway components and/or copy number loss in the NOTCH1 locus in 61% of the bladder cancer patients studied. Also, we demonstrated that Notch pathway genetic alterations do not often coexist with FGFR3, HRAS/KRAS or PIK3CA mutations and they correlate with worse survival.
Last, since there is a plethora of data indicating that retinoic acid signaling is implicated in the development and tumorigenesis of the urinary tract, we delved into the study of its role. Initially, we characterized the mutation in the hotspot S427F of the RXRα receptor, occurring in 5% of bladder cancer patients, as dominant-negative, given that ligand binding and therefore transcription activation is impaired while the heterodimerization capacity is retained. We also examined the effect of RXRα loss during chemical carcinogenesis in the mouse bladder and found that it leads to squamous metaplasia development. Based on the fact that Notch1 binds to the RXRA promoter, we concluded that the retinoic acid pathway acts downstream of Notch mediating part of its tumor suppressor role.
In conclusion, this research established the Notch signaling pathway as a tumor suppressor in the urinary tract and elucidated the mechanistic aspects of its role both in vitro by using human cancer cell lines and in vivo by using mouse models, paving the way for the development of novel targeted therapies with the aim of treating effectively bladder cancer.
