Activating mutations in CTNNB1 gene encoding β-catenin is encountered in approximately 30% of hepatocellular carcinoma (HCC) and in more than 80% of hepatoblastoma. In ApcΔhep model, the inventors unravel the biggest cluster of non-coding RNAs identified called the DLK1/DIO3 locus as the most significantly induced region in response to β-catenin activation regarding transcription of coding and non-coding elements. Using in vivo Crispr/cas9 strategy, the inventors were able to demonstrate that β-catenin and its cofactor TCF-4 directly bind on a WRE-containing site located upstream of Meg3 to create an active enhancer regulatory region engaged in chromatin remodeling in the direct vicinity of this binding site but also at distance
by long range DNA-DNA contacts to promote transcription of the entire locus. These
Crispr/cas9 constructs have also proved to be a valuable strategy to impair the locus expression in the murine models mimicking HCC and hepatoblastoma (ApcΔhep and β-catenin Exon3 tumors) but also in two cell lines with activating mutations in β-catenin encoding gene, the murine Hepa1-6 and human HuH6 cells. In transformed cells, it significantly impaired cell proliferation in vitro and HuH6 stemness capacities but also tumor progression in Hepa1-6 allografts. In mouse models, the locus editing during early steps of tumorigenesis decreased the proliferation of ApcΔhep preneoplastic hepatocytes but also those of ApcΔhep and β-catenin Exon3 tumor cell resulting in impairment of tumor size. In conclusion, the results demonstrate that disrupting the
-catenin/TCF-4 binding site located upstream of Meg3 in the DLK1/DIO3 locus represents a very interesting approach for the treatment of liver cancers.