Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano

9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug. The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation. On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects. In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent.

Role of (R)-9-hydroxystearic acid in zebrafish development

9-hydroxystearic acid (9-HSA) belongs to a class of lipid peroxidation products identified in several human and murine cell lines. These products are greatly diminished in tumors compared to normal tissues and their amount is inversely correlated with the malignancy of the tumor. 9-HSA activity has been tested in cancer cell lines, where it showed to act as a histone deacetylase 1 (HDAC1) inhibitor. In particular, in a colon cancer cell line (HT29), its administration resulted in an inhibition of proliferation together with an induction of differentiation. In this thesis the effect of (R)-9-hydroxystearic acid has been tested in vivo on cell proliferation and differentiation processes, in the early stages of zebrafish development. The final aim of this work was to elucidate the role of (R)-9-HSA in the control of cell differentiation and proliferation during normal development, in order to better understand its molecular control of cancerogenesis. The molecule has been administered via injection in the yolk of zebrafish embryos. The analysis of the histone acetylation pattern showed a hyperacetilation of histone H4 after treatment with the molecule, as detectable in HDAC1 mutants. (R)-9-HSA was also demonstrated to interfere with the signaling pathways that regulate proliferation and differentiation in zebrafish retina and hindbrain. This resulted in a reduction of proliferation in the hindbrain at 24 hours post injection (hpi), and in a hyperproliferation at 48 and 72 hpi in the retina, with a concomitant inhibition of differentiation. Finally, (R)-9-HSA effects were evident on proliferation of stem cell located in the ciliary marginal zone (CMZ) of the retina. The presence of ROS and 4-hydroxynoneal in the CMZ of wild-type embryos supports the hypothesis that oxidative stress could regulate stem cells fate in zebrafish retina.

Unbalanced R-loops and micronuclei induced by DNA topoisomerase I poisons in cancer cells

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.

R-loops and G4-structures: from DNA damage to innate immune response gene activation

Non-B DNA structures like R-loops and G-quadruplexes play a pivotal role in several cellular vital processes like DNA transcription regulation. Misregulation of said non-canonical DNA structures can often lead to genome instability, DNA damage, and, eventually, to the activation of an innate immune response. For such reasons they have been studied as adjuvants in anticancer therapies. Here we studied drugs targeting R-loops (Top1 poisons) and G4s (hydrazone derivatives) in order to observe their effects in terms of DNA damage induction and, subsequently, activation of innate immune response. We studied how non-cytotoxic doses of ampthotecin and LMP-776 impact on genome instability, are capable to induce DNA damage and micronuclei, and, eventually lead to an innate immune gene response via the cGAS/STING pathway. G-quadruplexes are another ubiquitous, non-canonical DNA structure, more abundant in telomeric regions, demonstrating a marked relation with the impairment of telomerase and the regulation of DNA replication and transcription. Furthermore, we investigated the properties of new-synthesized molecules belonging to the highly promising class of hydrazone derivatives, in terms of cytotoxicity, ability to stabilize G4-structures, induce DNA damage, and activate interferon-B production. Both Top1 poisons and G4-stabilizers possess several features that can be very useful in clinical applications, in light of their ability to stimulate innate immune response factors and exert a certain cell-killing power, plus they offer a broad and diverse range of treatment options in order to face a variety of patient treatment needs. It is for these very reasons that it is of uttermost importance that further studies are conducted on these compounds, in order to synthesize new and increasingly powerful and flexible ones, with fewer side effects to customize therapies on specific cancers’ and patients’ features.