Phd in MOLECULAR MEDICINE

Thesis title: Characterization of the mechanisms involved in KCASH oncosuppressors regulation

The Hedgehog (Hh) signaling pathway plays a pivotal role in the morphogenesis and tumorigenesis of various organs. The aberrant activation of a non-canonical Hh pathway and the onset of drug-resistant mutations in tumor cells, limit the use of standard Hh pathway inhibitors in cancer treatment, highlighting the need to develop new therapeutic strategies. One of these strategies could be based on the modulation of oncosuppressor KCASHs proteins. These proteins play an essential role in turning offthe Hh signalling, by binding Cullin 3 to form a E3 ubiquitin ligase complex which ubiquitinates and induces the degradation of the histone deacetylase HDAC1, blocking the activation of the main effector of the Hh pathway, Gli1 We have studied here the mechanisms of transcriptional regulation of KCASH2, analyzing the TATA-less KCASH2 proximal promoter and identifying key transcriptional regulators of this gene: Sp1 and p53. The data obtained suggest that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells were p53 functionality is lost). Moreover, to better characterize the modulation mechanism of KASH family we have searched for new KCASH interactors. In this work, we have demonstrated that KCTD1 is involved in the regulation of KCASH proteins and in the modulation of Hh pathway. Indeed, KCTD1 is able to bind both KCASH1 and KCASH2 proteins and inhibits their degradation, improving their negative effect on Hh activity., KCTD1 overexpression induces a decrease of HDAC1 protein levels and an increase of Gli1 acetylation, repressing Gli1 activity. The ability of KCTD1 to act as a modulator of both KCASH1 and KCASH2, unlike the paralogue KCTD15, highlights the crucial role of KCTD1 in the negative regulation of the Hedgehog pathway. Overall, the data presented in this thesis contribute to the characterization of the mechanisms modulating the KCASH family of inhibitors of the Hh pathway, suggesting new potential therapeutic strategies against the Hh dependent tumors.