Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Study of the role of the telomeric protein TRF2 in heterochromatin metabolism

TRF2 is a telomeric protein that binds directly to DNA and participates in shelterin complex formation, which covers telomeres and prevents linear extremities’ degradation, fusion and recombination. For several years, it has been known that TRF2 has a non-canonical extra-telomeric role. Indeed, it has been demonstrated that in TRF2-overexpressed cancer cells, it can regulate the expression of several oncogenes. Furthermore, it can bind genome-wide interstitial telomeric sequences (ITS), SatIII sequences and G-rich sequences. The latter are enriched in pericentromeric region, which contains nucleolar organizer regions, where rRNA genes are encoded. The ribosome pathway results highly altered in cancer cells, which require a high protein synthesis rate to continue proliferating. Considering the role of TRF2 in human cancers and its ability to bind extra-telomeric DNA sequences, we aimed to study the role of TRF2 in structural and functional regulation of nucleolus, investigating the possible role of TRF2 on rRNAs expression and ribosome biogenesis by identifying new molecular functions of TRF2 in tumours and new therapeutic strategies for anti-tumoral therapy. The role of TRF2 in nucleolar organization has emerged. In cancer cells, the overexpression of TRF2 leads to an overabundance of this protein in the nucleoplasm: some of them will bind to telomeric DNA, while the excess part will bind to other DNA regions with less affinity. We demonstrated that TRF2 can affect the ribosomal pathway, not by acting on rDNA gene transcription, but by directly binding to specific rRNAs. Moreover, the overexpression of TRF2 is associated with an increased protein synthesis rate, which is consistent with its pro-oncogenic role. Furthermore, we discovered an antagonistic role of TRF2 over-expression concernign CX5461, a clinically employed compound which acts as ribosomal pathway inhibitor. Additionally, we discovered a new mechanism of action of a naphthalene diimide dimer that, through a different mechanism than CX5461, severely impairs protein synthesis, exhibiting high cytotoxicity in different cancer cell lines at nanomolar concentrations. This newly identified role of TRF2 in the regulation of the ribosomal pathway in cancers may open novel targeted therapies to block cancer progression and development. In summary, this work establishes a new role for TRF2 at nucleoli, where it acts directly on ribosome assembly and protein synthesis and identifies a new potential drug that acts on this pathway to fight cancer development.