Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and one of the most difficult to treat. HCCs, in fact, often develop on severe pre-existing chronic liver diseases, in particular fibrosis or cirrhosis, that impair organ function and make inappropriate any potentially curative approach. Several studies suggested the high therapeutic potential of master regulators of hepatocyte differentiation belonging to the LETF family, mainly HNF4, HNF1 and HNF6, whose loss represents a common feature of advanced-stage HCC. Moreover, preclinical data showed that the transduction of these proteins in vivo in mouse models, prevents tumor formation and induces regression of established tumors. However these approaches, although promising, need to take in account the micro-environmental cues that can influence the effectiveness of therapies. Our recent data, in particular, indicated that the efficacy of HNF4 gene delivery can be limited by TGF, a cytokine known to induce tumor progression, angiogenesis and epithelial-to mesenchymal transition. These studies demonstrated that TGFβ impairs tumor suppressor activity of exogenous HNF4 through the inactivation of the kinase GSK-3β, which is needed for both HNF4 DNA binding and phosphorylation. Taking into account all these observations, the aim of this work was to develop new molecular tools, insensitive to the presence of TGFβ in the tumor microenvironment, for the gene therapy of HCC, based on the restoration of HNF expression/activity. On one hand, we attempted the characterization of the GSK-3β-mediated phosphorylation on HNF4α protein in order to develop HNF4α mutant proteins insensitive to TGFβ-induced inactivation. At the same time, we investigated the potential use of HNF1α and HNF6, analyzing their possible resistance to the TGFβ-induced impairment. First, we demonstrated that HNF4α is a direct target of phosphorylation by GSK-3β. The residues involved in this phosphorylation were predicted by in silico studies and mutated to produce phosphomimetic mutants. After the assessment of the in vivo functionality of mutant proteins we demonstrated that the HNF4α protein, mutated in three residues (Ser143, Thr422 and Ser426), was made resistant to the inactivation by both a chemical inhibitor of GSK-3β kinase and TGFβ, indicating the involvement of the identified residues i) in the GSK-3β -induced phosphorylation of HNF4α and ii) in the TGFβ- induced HNF4α functional inactivation. These results support the potential of our triple mutant as therapeutic tool for HCC treatment. Next, we found that TGFβ was also able to override in vivo transcriptional activity of HNF1α and HNF6. However, no impairment of their DNA binding activity was observed, indicating that the mechanism involved in their functional inactivation is different from that observed for the HNF4α protein. In fact, we demonstrated that TGFβ induced a chromatin remodeling of HNF1α target gene promoters, indicative of a “closed” and inactive chromatin state. In particular, we observed the early loss of H3 acetylation, correlated with the displacement of CBP/p300 acetyl transferase from HNF1α binding sites. This result was confirmed by a reduced physical interaction of HNF1α protein with CBP/p300. Collectively, data obtained in this work unveiled new mechanisms involved in the dominance of TGFβ over transcriptional activity of HNFs and identified potential therapeutic tools for the molecular therapy of HCC.
Developing new tools for gene therapy of HCC / Bisceglia, Francesca. - (2017 Feb 27).
|Titolo:||Developing new tools for gene therapy of HCC|
|Data di discussione:||27-feb-2017|
|Appartiene alla tipologia:||07a Tesi di Dottorato|