Hepatocyte identity and zonal position determine tumourigenic potential of mutant β-catenin

<title>Abstract</title> Oncogenic mutations in otherwise normal tissue are common in many adult tissues ^1,2 . This suggests multiple events need to converge to drive tumourigenesis and that many processes such as tissue differentiation may be protective against carcinogenesis. Within the liver Wnt/β-catenin signalling maintains zonal differentiation during liver homeostasis ^3,4 . However, the <italic>CTNNB1</italic> oncogene—encoding β-catenin—is also frequently mutated in hepatocellular carcinoma, resulting in aberrant Wnt signalling that promotes cell growth ^5,6 . Here we investigated the antagonistic interplay between Wnt-driven growth and differentiation in zonal hepatocyte populations during liver tumorigenesis. We found that β-catenin mutants transiently stimulated growth in zone-1 and -2 hepatocytes within the Wnt-low region of the liver lobule – before inducing a differentiated zone-3 fate. One key feature lacking within the differentiated zone-3 hepatocytes that is often upregulated in human cancer was MYC which can synergise with β-catenin to initiate HCC in mice. When mutant β-catenin and <italic>MYC</italic> were expressed cell-agnostically across the liver lobule, a subset of mutant hepatocytes were proliferative and tumorigenic. These exhibited a selective upregulation of the zone-2 factors IGFBP2, cyclin D1, and mTOR and a reduction in the levels of Wnt signalling when compared to non-proliferative Wnt-high single cell clones. Inhibition of mTOR signalling with Rapamycin blocked tumourigenesis. Furthermore, restricting β-catenin and <italic>MYC</italic> mutations with Lgr5-Cre to zone-3 hepatocytes suppressed tumourigenesis. This was associated with high Wnt signalling and the lack of zone-2 factors permissive to growth. Importantly other oncogenic-drivers could promote tumourigenesis from zone-3 hepatocytes but tumours that arose had all lost their zone 3 features. Therefore we propose that zonal identity dictates hepatocyte susceptibility to Wnt-driven tumorigenesis and that escaping Wnt-induced differentiation is essential for liver cancer.