Estudio de "G-quadruplexes" del ADN en cáncer colorrectal y su uso como diana terapéutica

Abstract

DNA guanine quadruplexes (G4s) are non-canonical structures formed through self-recognition of four guanines into stacked tetrads. G4s are highly prevalent at regulatory genomic regions. Considerable evidence has linked G4 formation with key biological processes ranging from telomere maintenance and transcription to genome instability and cancer. In particular, colorectal cancer (CRC) is the third most diagnosed cancer and constitutes the second leading cause of cancer death worldwide. New treatment options for CRC are required. To mimic the progression of CRC, we established a cellular model including non-tumoral, primary tumor and metastatic stages. Despite helicases involved in unwinding of G4s were overexpressed in CRC, stabilization of G4s and induction of DNA damage increased along CRC progression, both at G0/G1 and S phases. We identified a link between the presence of G4s and the accumulation of double-strand breaks in their vicinity. The folding status of G4s played a role in the abnormal gene expression of CRC-relevant genes such as CMYC. The G4 harbored in its promoter region did not contain any mutation. Several well-known G4 ligands induced cytotoxicity, but lacked selectivity for tumoral cells. In addition, we screened in CRC the antitumoral activity of several naphthalene-diimides (NDIs), a class of G4 ligands. We identified the leading compound T5 with a potent and selective inhibition of tumoral cell growth by high-affinity binding to G4s present in ribosomal DNA, thereby impairing RNA polymerase I (Pol I) elongation. Consequently, T5 induced a rapid inhibition of Pol I transcription, nucleolus disruption, proteasomedependent Pol I catalytic subunit A degradation, and autophagy. Moreover, we attributed the higher selectivity of carbohydrate-conjugated T5 for tumoral cells to its preferential uptake through the overexpressed glucose transporter 1. We succinctly demonstrated that T5 could be explored as a therapeutic agent in a patient cohort with CRC. Furthermore, we screened in CRC the antitumoral potential of several natural phenolic compounds. We selected gallic acid (GA) as candidate in terms of potency and selectivity. We reported on the role of GA as a G4 ligand explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared some effects with other established G4 ligands such as cell cycle arrest, nucleolar stress, and induction of DNA damage and autophagy. We further confirmed antitumoral and G4-stabilizing properties of GA in vivo using a xenograft model of CRC. Moreover, we concisely demonstrated that treatment with GA could be exploitable in a patient cohort with CRC. Finally, we generated a library of nanobodies targeting G4s through in vitro immunization. Nanobodies derive from heavy chain camelid antibodies by recombinant DNA technology. Although nanobodies displayed cross-reactivity with different G4 structures, specific CMYC G4- targeting nanobodies were enriched after phage-display biopanning. Overall, in this PhD thesis, we have analyzed G4 involvement in colorectal carcinogenesis, and we have investigated the therapeutic potential of several G4 ligands in CRC. We have disclosed a mode of action for NDIs that involves ribosomal G4s targeting, and that GA affects gene expression by interaction with G4s both in vitro and in vivo.